Compositions and Methods for the Treatment of Tumor of Hematopoietic Origin

ABSTRACT

The present invention is directed to compositions of matter useful for the treatment of hematopoietic tumor in mammals and to methods of using those compositions of matter for the same.

RELATED APPLICATIONS

This application is a continuation-in-part of, and claims priority under35 USC § 120 to U.S. application Ser. No. 11/461,752, filed Aug. 1,2006, which claims priority under 35 USC § 120 to both, PCT ApplicationNo. PCT/US2004/038262, filed Nov. 16, 2004, and also to U.S. applicationSer. No. 10/989,826, filed Nov. 16, 2004, both of which claim priorityunder USC § 119 to U.S. Provisional Application 60/520,842, filed Nov.17, 2003, and also to 60/532,426, filed Dec. 24, 2003, and wherein thepresent application is also a continuation-in-part of, and claimspriority under 35 USC § 120 to both PCT/US2005/018,829, filed May 31,2005, and also to U.S. application Ser. No. 11/141,344, filed May 31,2005, both of which claim priority under USC §119 to U.S. ProvisionalApplication 60/576,517, filed Jun. 1, 2004, and 60/616,098, filed Oct.5, 2004.

FIELD OF THE INVENTION

The present invention is directed to compositions of matter useful forthe treatment of hematopoietic tumor in mammals and to methods of usingthose compositions of matter for the same.

BACKGROUND OF THE INVENTION

Malignant tumors (cancers) are the second leading cause of death in theUnited States, after heart disease (Boring et al., CA Cancel J. Clin.43:7 (1993)). Cancer is characterized by the increase in the number ofabnormal, or neoplastic, cells derived from a normal tissue whichproliferate to form a tumor mass, the invasion of adjacent tissues bythese neoplastic tumor cells, and the generation of malignant cellswhich eventually spread via the blood or lymphatic system to regionallymph nodes and to distant sites via a process called metastasis. In acancerous state, a cell proliferates under conditions in which normalcells would not grow. Cancer manifests itself in a wide variety offorms, characterized by different degrees of invasiveness andaggressiveness.

Cancers which involve cells generated during hematopoiesis, a process bywhich cellular elements of blood, such as lymphocytes, leukocytes,platelets, erythrocytes and natural killer cells are generated arereferred to as hematopoictic cancers. Lymphocytes which can be found inblood and lymphatic tissue and are critical for immune response arecategorized into two main classes of lymphocytes: B lymphocytes (Bcells) and T lymphocytes (T cells), which mediate humoral and cellmediated immunity, respectively.

B cells mature within the bone marrow and leave the marrow expressing anantigen-binding antibody on their cell surface. When a naive B cellfirst encounters the antigen for which its membrane-bound antibody isspecific, the cell begins to divide rapidly and its progenydifferentiate into memory B cells and effector cells called “plasmacells”. Memory B cells have a longer life span and continue to expressmembrane-bound antibody with the same specificity as the original parentcell. Plasma cells do not produce membrane-bound antibody but insteadproduce the antibody in a form that can be secreted. Secreted antibodiesare the major effector molecule of humoral immunity.

T cells mature within the thymus which provides an environment for theproliferation and differentiation of immature T cells. During T cellmaturation, the T cells undergo the gene rearrangements that produce theT-cell receptor and the positive and negative selection which helpsdetermine the cell-surface phenotype of the mature T cell.Characteristic cell surface markers of mature T cells are the CD3:T-cellreceptor complex and one of the coreceptors, CD4 or CD8.

In attempts to discover effective cellular targets for cancer therapy,researchers have sought to identify transmembrane or otherwisemembrane-associated polypeptides that are specifically expressed on thesurface of one or more particular type(s) of cancer cell as compared toon one or more normal non-cancerous cell(s). Often, suchmembrane-associated polypeptides are more abundantly expressed on thesurface of the cancer cells as compared to on the surface of thenon-cancerous cells. The identification of such tumor-associated cellsurface antigen polypeptides has given rise to the ability tospecifically target cancer cells for destruction via antibody-basedtherapies. In this regard, it is noted that antibody-based therapy hasproved very effective in the treatment of certain cancers. For example,HERCEPTIN® and RITUXAN® (both from Genentech Inc., South San Francisco,Calif.) are antibodies that have been used successfully to treat breastcancer and non-Hodgkin's lymphoma, respectively. More specifically,HERCEPTIN® is a recombinant DNA-derived humanized monoclonal antibodythat selectively binds to the extracellular domain of the humanepidermal growth factor receptor 2 (HER2) proto-oncogene. HER2 proteinoverexpression is observed in 25-30% of primary breast cancers. RITUXAN®is a genetically engineered chimeric murine/human monoclonal antibodydirected against the CD20 antigen found on the surface of normal andmalignant B lymphocytes. Both these antibodies are recombinantlyproduced in CHO cells.

In other attempts to discover effective cellular targets for cancertherapy, researchers have sought to identify (1) non-membrane-associatedpolypeptides that are specifically produced by one or more particulartype(s) of cancer cell(s) as compared to by one or more particulartype(s) of non-cancerous normal cell(s), (2) polypeptides that areproduced by cancer cells at an expression level that is significantlyhigher than that of one or more normal non-cancerous cell(s), or (3)polypeptides whose expression is specifically limited to only a single(or very limited number of different) tissue type(s) in both thecancerous and non-cancerous state (e.g., normal prostate and prostatetumor tissue). Such polypeptides may remain intracellularly located ormay be secreted by the cancer cell. Moreover, such polypeptides may beexpressed not by the cancer cell itself, but rather by cells whichproduce and/or secrete polypeptides having a potentiating orgrowth-enhancing effect on cancer cells. Such secreted polypeptides areoften proteins that provide cancer cells with a growth advantage overnormal cells and include such things as, for example, angiogenicfactors, cellular adhesion factors, growth factors, and the like.Identification of antagonists of such non-membrane associatedpolypeptides would be expected to serve as effective therapeutic agentsfor the treatment of such cancers. Furthermore, identification of theexpression pattern of such polypeptides would be useful for thediagnosis of particular cancers in mammals.

Despite the above identified advances in mammalian cancer therapy, thereis a great need for additional therapeutic agents capable of detectingthe presence of tumor in a mammal and for effectively inhibitingneoplastic cell growth, respectively. Accordingly, it is an objective ofthe present invention to identify polypeptides, cellmembrane-associated, secreted or intracellular polypeptides whoseexpression is specifically limited to only a single (or very limitednumber of different) tissue type(s), hematopoietic tissues, in both acancerous and non-cancerous state, and to use those polypeptides, andtheir encoding nucleic acids, to produce compositions of matter usefulin the therapeutic treatment detection of hematopoietic cancer inmammals.

SUMMARY OF THE INVENTION A. Embodiments

In the present specification, Applicants describe for the first time theidentification of various cellular polypeptides (and their encodingnucleic acids or fragments thereof) which are specifically expressed byboth tumor and normal cells of a specific cell type, for example cellsgenerated during hematopoiesis, i.e. lymphocytes, leukocytes,erythrocytes and platelets. All of the above polypeptides are hereinreferred to as Tumor Antigens of Hematopoietic Origin polypeptides(“TAHO” polypeptides) and are expected to serve as effective targets forcancer therapy in mammals.

Accordingly, in one embodiment of the present invention, the inventionprovides an isolated nucleic acid molecule having a nucleotide sequencethat encodes a tumor antigen of hematopoietic origin polypeptide (a“TAHO” polypeptide) or fragment thereof.

In certain aspects, the isolated nucleic acid molecule comprises anucleotide sequence having at least about 80% nucleic acid sequenceidentity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%nucleic acid sequence identity, to (a) a DNA molecule encoding afull-length TAHO polypeptide having an amino acid sequence as disclosedherein, a TAHO polypeptide amino acid sequence lacking the signalpeptide as disclosed herein, an extracellular domain of a transmembraneTAHO polypeptide, with or without the signal peptide, as disclosedherein or any other specifically defined fragment of a full-length TAHOpolypeptide amino acid sequence as disclosed herein, or (b) thecomplement of the DNA molecule of (a).

In other aspects, the isolated nucleic acid molecule comprises anucleotide sequence having at least about 80% nucleic acid sequenceidentity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%nucleic acid sequence identity, to (a) a DNA molecule comprising thecoding sequence of a full-length TAHO polypeptide cDNA as disclosedherein, the coding sequence of a TAHO polypeptide lacking the signalpeptide as disclosed herein, the coding sequence of an extracellulardomain of a transmembrane TAHO polypeptide, with or without the signalpeptide, as disclosed herein or the coding sequence of any otherspecifically defined fragment of the full-length TAHO polypeptide aminoacid sequence as disclosed herein, or (b) the complement of the DNAmolecule of (a).

In further aspects, the invention concerns an isolated nucleic acidmolecule comprising a nucleotide sequence having at least about 80%nucleic acid sequence identity, alternatively at least about 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% nucleic acid sequence identity, to (a) a DNAmolecule that encodes the same mature polypeptide encoded by thefull-length coding region of any of the human protein cDNAs depositedwith the ATCC as disclosed herein, or (b) the complement of the DNAmolecule of (a).

Another aspect of the invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence encoding a TAHO polypeptidewhich is either transmembrane domain-deleted or transmembranedomain-inactivated, or is complementary to such encoding nucleotidesequence, wherein the transmembrane domain(s) of such polypeptide(s) aredisclosed herein. Therefore, soluble extracellular domains of the hereindescribed TAHO polypeptides are contemplated.

In other aspects, the present invention is directed to isolated nucleicacid molecules which hybridize to (a) a nucleotide sequence encoding aTAHO polypeptide having a full-length amino acid sequence as disclosedherein, a TAHO polypeptide amino acid sequence lacking the signalpeptide as disclosed herein, an extracellular domain of a transmembraneTAHO polypeptide, with or without the signal peptide, as disclosedherein or any other specifically defined fragment of a full-length TAHOpolypeptide amino acid sequence as disclosed herein, or (b) thecomplement of the nucleotide sequence of (a). In this regard, anembodiment of the present invention is directed to fragments of afull-length TAHO polypeptide coding sequence, or the complement thereof,as disclosed herein, that may find use as, for example, hybridizationprobes useful as, for example, detection probes, antisenseoligonucleotide probes, or for encoding fragments of a full-length TAHOpolypeptide that may optionally encode a polypeptide comprising abinding site for an anti-TAHO polypeptide antibody, a TAHO bindingoligopeptide or other small organic molecule that binds to a TAHOpolypeptide. Such nucleic acid fragments are usually at least about 5nucleotides in length, alternatively at least about 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110,115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180,185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440,450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580,590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720,730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860,870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000nucleotides in length, wherein in this context the term “about” meansthe referenced nucleotide sequence length plus or minus 10% of thatreferenced length. It is noted that novel fragments of a TAHOpolypeptide-encoding nucleotide sequence may be determined in a routinemanner by aligning the TAHO polypeptide-encoding nucleotide sequencewith other known nucleotide sequences using any of a number of wellknown sequence alignment programs and determining which TAHOpolypeptide-encoding nucleotide sequence fragment(s) are novel. All ofsuch novel fragments of TAHO polypeptide-encoding nucleotide sequencesare contemplated herein. Also contemplated are the TAHO polypeptidefragments encoded by these nucleotide molecule fragments, preferablythose TAHO polypeptide fragments that comprise a binding site for ananti-TAHO antibody, a TAHO binding oligopeptide or other small organicmolecule that binds to a TAHO polypeptide.

In another embodiment, the invention provides isolated TAHO polypeptidesencoded by any of the isolated nucleic acid sequences hereinaboveidentified.

In a certain aspect, the invention concerns an isolated TAHOpolypeptide, comprising an amino acid sequence having at least about 80%amino acid sequence identity, alternatively at least about 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% amino acid sequence identity, to a TAHOpolypeptide having a full-length amino acid sequence as disclosedherein, a TAHO polypeptide amino acid sequence lacking the signalpeptide as disclosed herein, an extracellular domain of a transmembraneTAHO polypeptide protein, with or without the signal peptide, asdisclosed herein, an amino acid sequence encoded by any of the nucleicacid sequences disclosed herein or any other specifically definedfragment of a full-length TAHO polypeptide amino acid sequence asdisclosed herein.

In a further aspect, the invention concerns an isolated TAHO polypeptidecomprising an amino acid sequence having at least about 80% amino acidsequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%amino acid sequence identity, to an amino acid sequence encoded by anyof the human protein cDNAs deposited with the ATCC as disclosed herein.

In a specific aspect, the invention provides an isolated TAHOpolypeptide without the N-terminal signal sequence and/or without theinitiating methionine and is encoded by a nucleotide sequence thatencodes such an amino acid sequence as hereinbefore described. Processesfor producing the same are also herein described, wherein thoseprocesses comprise culturing a host cell comprising a vector whichcomprises the appropriate encoding nucleic acid molecule underconditions suitable for expression of the TAHO polypeptide andrecovering the TAHO polypeptide from the cell culture.

Another aspect of the invention provides an isolated TAHO polypeptidewhich is either transmembrane domain-deleted or transmembranedomain-inactivated. Processes for producing the same are also hereindescribed, wherein those processes comprise culturing a host cellcomprising a vector which comprises the appropriate encoding nucleicacid molecule under conditions suitable for expression of the TAHOpolypeptide and recovering the TAHO polypeptide from the cell culture.

In other embodiments of the present invention, the invention providesvectors comprising DNA encoding any of the herein describedpolypeptides. Host cells comprising any such vector are also provided.By way of example, the host cells may be CHO cells, E. coli cells, oryeast cells. A process for producing any of the herein describedpolypeptides is further provided and comprises culturing host cellsunder conditions suitable for expression of the desired polypeptide andrecovering the desired polypeptide from the cell culture.

In other embodiments, the invention provides isolated chimericpolypeptides comprising any of the herein described TAHO polypeptidesfused to a heterologous (non-TAHO) polypeptide. Example of such chimericmolecules comprise any of the herein described TAHO polypeptides fusedto a heterologous polypeptide such as, for example, an epitope tagsequence or a Fc region of an immunoglobulin.

In another embodiment, the invention provides an antibody which binds,preferably specifically, to any of the above or below describedpolypeptides. Optionally, the antibody is a monoclonal antibody,antibody fragment, chimeric antibody, humanized antibody, single-chainantibody or antibody that competitively inhibits the binding of ananti-TAHO polypeptide antibody to its respective antigenic epitope.Antibodies of the present invention may optionally be conjugated to agrowth inhibitory agent or cytotoxic agent such as a toxin, including,for example, a maytansinoid or calicheamicin, an antibiotic, aradioactive isotope, a nucleolytic enzyme, or the like. The antibodiesof the present invention may optionally be produced in CHO cells orbacterial cells and preferably induce death of a cell to which theybind. For detection purposes, the antibodies of the present inventionmay be detectably labeled, attached to a solid support, or the like.

In other embodiments of the present invention, the invention providesvectors comprising DNA encoding any of the herein described antibodies.Host cell comprising any such vector are also provided. By way ofexample, the host cells may be CHO cells, E. coli cells, or yeast cells.A process for producing any of the herein described antibodies isfurther provided and comprises culturing host cells under conditionssuitable for expression of the desired antibody and recovering thedesired antibody from the cell culture.

In another embodiment, the invention provides oligopeptides (“TAHObinding oligopeptides”) which bind, preferably specifically, to any ofthe above or below described TAHO polypeptides. Optionally, the TAHObinding oligopeptides of the present invention may be conjugated to agrowth inhibitory agent or cytotoxic agent such as a toxin, including,for example, a maytansinoid or calicheamicin, an antibiotic, aradioactive isotope, a nucleolytic enzyme, or the like. The TAHO bindingoligopeptides of the present invention may optionally be produced in CHOcells or bacterial cells and preferably induce death of a cell to whichthey bind. For detection purposes, the TAHO binding oligopeptides of thepresent invention may be detectably labeled, attached to a solidsupport, or the like.

In other embodiments of the present invention, the invention providesvectors comprising DNA encoding any of the herein described TAHO bindingoligopeptides. Host cell comprising any such vector are also provided.By way of example, the host cells may be CHO cells, E. coli cells, oryeast cells. A process for producing any of the herein described TAHObinding oligopeptides is further provided and comprises culturing hostcells under conditions suitable for expression of the desiredoligopeptide and recovering the desired oligopeptide from the cellculture.

In another embodiment, the invention provides small organic molecules(“TAHO binding organic molecules”) which bind, preferably specifically,to any of the above or below described TAHO polypeptides. Optionally,the TAHO binding organic molecules of the present invention may beconjugated to a growth inhibitory agent or cytotoxic agent such as atoxin, including, for example, a maytansinoid or calicheamicin, anantibiotic, a radioactive isotope, a nucleolytic enzyme, or the like.The TAHO binding organic molecules of the present invention preferablyinduce death of a cell to which they bind. For detection purposes, theTAHO binding organic molecules of the present invention may bedetectably labeled, attached to a solid support, or the like.

In a still further embodiment, the invention concerns a composition ofmatter comprising a TAHO polypeptide as described herein, a chimericTAHO polypeptide as described herein, an anti-TAHO antibody as describedherein, a TAHO binding oligopeptide as described herein, or a TAHObinding organic molecule as described herein, in combination with acarrier. Optionally, the carrier is a pharmaceutically acceptablecarrier.

In yet another embodiment, the invention concerns an article ofmanufacture comprising a container and a composition of matter containedwithin the container, wherein the composition of matter may comprise aTAHO polypeptide as described herein, a chimeric TAHO polypeptide asdescribed herein, an anti-TAHO antibody as described herein, a TAHObinding oligopeptide as described herein, or a TAHO binding organicmolecule as described herein. The article may further optionallycomprise a label affixed to the container, or a package insert includedwith the container, that refers to the use of the composition of matterfor the therapeutic treatment.

Another embodiment of the present invention is directed to the use of aTAHO polypeptide as described herein, a chimeric TAHO polypeptide asdescribed herein, an anti-TAHO polypeptide antibody as described herein,a TAHO binding oligopeptide as described herein, or a TAHO bindingorganic molecule as described herein, for the preparation of amedicament useful in the treatment of a condition which is responsive tothe TAHO polypeptide, chimeric TAHO polypeptide, anti-TAHO polypeptideantibody, TAHO binding oligopeptide, or TAHO binding organic molecule.

B. Further Additional Embodiments

In yet further embodiments, the invention is directed to the followingset of potential claims for this application:

1. Isolated nucleic acid having a nucleotide sequence that has at least80% nucleic acid sequence identity to:

(a) a DNA molecule encoding the amino acid sequence selected from thegroup consisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6);

(b) a DNA molecule encoding the amino acid sequence selected from thegroup consisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lacking itsassociated signal peptide;

(c) a DNA molecule encoding an extracellular domain of the polypeptidehaving the amino acid selected from the group consisting of the aminoacid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) andFIG. 6 (SEQ ID NO: 6), with its associated signal peptide;

(d) a DNA molecule encoding an extracellular domain of the polypeptidehaving the amino acid selected from the group consisting of the aminoacid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) andFIG. 6 (SEQ ID NO: 6), lacking its associated signal peptide;

(e) the nucleotide sequence selected from the group consisting of thenucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3)and FIG. 5 (SEQ ID NO: 5);

(f) the full-length coding region of the nucleotide sequence selectedfrom the group consisting of the nucleotide sequence shown in FIG. 1(SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or

(g) the complement of (a), (b), (c), (d), (e) or (f).

2. Isolated nucleic acid having:

(a) a nucleotide sequence that encodes the amino acid sequence selectedfrom the group consisting of the amino acid sequence shown in FIG. 2(SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6);

(b) a nucleotide sequence that encodes the amino acid sequence selectedfrom the group consisting of the amino acid sequence shown in FIG. 2(SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lackingits associated signal peptide;

(c) a nucleotide sequence that encodes an extracellular domain of thepolypeptide having the amino acid selected from the group consisting ofthe amino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ IDNO: 4) and FIG. 6 (SEQ ID NO: 6), with its associated signal peptide;

(d) a nucleotide sequence that encodes an extracellular domain of thepolypeptide having the amino acid selected from the group consisting ofthe amino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ IDNO: 4) and FIG. 6 (SEQ ID NO: 6), lacking its associated signal peptide;

(e) the nucleotide sequence selected from the group consisting of thenucleotide sequence shown in FIG. 1 (SEQ ID NO: 1) and FIG. 3 (SEQ IDNO:3);

(f) the full-length coding region of the nucleotide sequence selectedfrom the group consisting of the nucleotide sequence shown in FIG. 1(SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or

(g) the complement of (a), (b), (c), (d), (e) or (f).

3. Isolated nucleic acid that hybridizes to:

(a) a nucleic acid that encodes the amino acid sequence selected fromthe group consisting of the amino acid sequence shown in FIG. 2 (SEQ IDNO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6);

(b) a nucleic acid that encodes the amino acid sequence selected fromthe group consisting of the amino acid sequence shown in FIG. 2 (SEQ IDNO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lacking itsassociated signal peptide;

(c) a nucleic acid that encodes an extracellular domain of thepolypeptide having the amino acid sequence selected from the groupconsisting of the amino acid sequence shown FIG. 2 (SEQ ID NO: 2), FIG.4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), with its associated signalpeptide;

(d) a nucleic acid that encodes an extracellular domain of thepolypeptide having the amino acid sequence selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2),FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lacking its associatedsignal peptide;

(e) the nucleotide sequence selected from the group consisting of thenucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3)and FIG. 5 (SEQ ID NO: 5);

(f) the full-length coding region of the nucleotide sequence selectedfrom the group consisting of the nucleotide sequence shown in FIG. 1(SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or

(g) the complement of (a), (b), (c), (d), (e) or (f).

4. The nucleic acid of claim 3, wherein the hybridization occurs understringent conditions.

5. The nucleic acid of claim 3 which is at least about 5 nucleotides inlength.

6. An expression vector comprising the nucleic acid of claim 1, 2 or 3.

7. The expression vector of claim 6, wherein said nucleic acid isoperably linked to control sequences recognized by a host celltransformed with the vector.

8. A host cell comprising the expression vector of claim 7.

9. The host cell of claim 8 which is a CHO cell, an E. coli cell or ayeast cell.

10. A process for producing a polypeptide comprising culturing the hostcell of claim 8 under conditions suitable for expression of saidpolypeptide and recovering said polypeptide from the cell culture.

11. An isolated polypeptide having at least 80% amino acid sequenceidentity to:

(a) the polypeptide having the amino acid sequence selected from thegroup consisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6);

(b) the polypeptide having the amino acid sequence selected from thegroup consisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lacking itsassociated signal peptide;

(c) an extracellular domain of the polypeptide having the amino acidsequence selected from the group consisting of the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ IDNO: 6), with its associated signal peptide;

(d) an extracellular domain of the polypeptide having the amino acidsequence selected from the group consisting of the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ IDNO: 6) lacking its associated signal peptide;

(e) a polypeptide encoded by the nucleotide sequence selected from thegroup consisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or

(f) a polypeptide encoded by the full-length coding region of thenucleotide sequence selected from the group consisting of the nucleotidesequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5(SEQ ID NO: 5).

12. An isolated polypeptide having:

(a) the amino acid sequence selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO:4) and FIG. 6 (SEQ ID NO: 6);

(b) the amino acid sequence selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO:4) and FIG. 6 (SEQ ID NO: 6), lacking its associated signal peptidesequence;

(c) an amino acid sequence of an extracellular domain of the polypeptideselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6),with its associated signal peptide sequence;

(d) an amino acid sequence of an extracellular domain of the polypeptideselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6),lacking its associated signal peptide sequence;

(e) an amino acid sequence encoded by the nucleotide sequence selectedfrom the group consisting of the nucleotide sequence shown in FIG. 1(SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO:5); or

(f) an amino acid sequence encoded by the full-length coding region ofthe nucleotide sequence selected from the group consisting of thenucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3)and FIG. 5 (SEQ ID NO: 5).

13. A chimeric polypeptide comprising the polypeptide of claim 11 or 12fused to a heterologous polypeptide.

14. The chimeric polypeptide of claim 13, wherein said heterologouspolypeptide is an epitope tag sequence or an Fc region of animmunoglobulin.

15. An isolated antibody that binds to a polypeptide having at least 80%amino acid sequence identity to:

(a) the polypeptide having the amino acid sequence selected from thegroup consisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6);

(b) the polypeptide selected from the group consisting of the amino acidsequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG.6 (SEQ ID NO: 6), lacking its associated signal peptide;

(c) an extracellular domain of the polypeptide having the amino acidsequence selected from the group consisting of the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ IDNO: 6), with its associated signal peptide;

(d) an extracellular domain of the polypeptide having the amino acidsequence selected from the group consisting of the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ IDNO: 6), lacking its associated signal peptide;

(e) a polypeptide encoded by the nucleotide sequence selected from thegroup consisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or

(f) a polypeptide encoded by the full-length coding region of thenucleotide sequence selected from the group consisting of the nucleotidesequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5(SEQ ID NO: 5).

16. An isolated antibody that binds to a polypeptide having:

(a) the amino acid sequence selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO:4) and FIG. 6 (SEQ ID NO: 6);

(b) the amino acid sequence selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO:4) and FIG. 6 (SEQ ID NO: 6), lacking its associated signal peptidesequence;

(c) an amino acid sequence of an extracellular domain of the polypeptideselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6),with its associated signal peptide sequence;

(d) an amino acid sequence of an extracellular domain of the polypeptideselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6),lacking its associated signal peptide sequence;

(e) an amino acid sequence encoded by the nucleotide sequence selectedfrom the group consisting of the nucleotide sequence shown in FIG. 1(SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or

(f) an amino acid sequence encoded by the full-length coding region ofthe nucleotide sequence selected from the group consisting of thenucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3)and FIG. 5 (SEQ ID NO: 5).

17. The antibody of claim 15 or 16 which is a monoclonal antibody.

18. The antibody of claim 15 or 16 which is an antibody fragment.

19. The antibody of claim 15 or 16 which is a chimeric or a humanizedantibody.

20. The antibody of claim 15 or 16 which is conjugated to a growthinhibitory agent.

21. The antibody of claim 15 or 16 which is conjugated to a cytotoxicagent.

22. The antibody of claim 21, wherein the cytotoxic agent is selectedfrom the group consisting of toxins, antibiotics, radioactive isotopesand nucleolytic enzymes.

23. The antibody of claim 21, wherein the cytotoxic agent is a toxin.

24. The antibody of claim 23, wherein the toxin is selected from thegroup consisting of maytansinoid and calicheamicin.

25. The antibody of claim 23, wherein the toxin is a maytansinoid.

26. The antibody of claim 15 or 16 which is produced in bacteria.

27. The antibody of claim 15 or 16 which is produced in CHO cells.

28. The antibody of claim 15 or 16 which induces death of a cell towhich it binds.

29. The antibody of claim 15 or 16 which is detectably labeled.

30. An isolated nucleic acid having a nucleotide sequence that encodesthe antibody of claim 15 or 16.

31. An expression vector comprising the nucleic acid of claim 30operably linked to control sequences recognized by a host celltransformed with the vector.

32. A host cell comprising the expression vector of claim 31.

33. The host cell of claim 32 which is a CHO cell, an E. coli cell or ayeast cell.

34. A process for producing an antibody comprising culturing the hostcell of claim 32 under conditions suitable for expression of saidantibody and recovering said antibody from the cell culture.

35. An isolated oligopeptide that binds to a polypeptide having at least80% amino acid sequence identity to:

(a) the polypeptide having the amino acid sequence selected from thegroup consisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6);

(b) the polypeptide having the amino acid sequence selected from thegroup consisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lacking itsassociated signal peptide;

(c) an extracellular domain of the polypeptide having the amino acidsequence selected from the group consisting of the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ IDNO: 6), with its associated signal peptide;

(d) an extracellular domain of the polypeptide having the amino acidsequence selected from the group consisting of the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ IDNO: 6), lacking its associated signal peptide;

(e) a polypeptide encoded by the nucleotide sequence selected from thegroup consisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or

(f) a polypeptide encoded by the full-length coding region of thenucleotide sequence selected from the group consisting of the nucleotidesequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5(SEQ ID NO: 5).

36. An isolated oligopeptide that binds to a polypeptide having:

(a) the amino acid sequence selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO:4) and FIG. 6 (SEQ ID NO: 6);

(b) the amino acid sequence selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO:4) and FIG. 6 (SEQ ID NO: 6), lacking its associated signal peptidesequence;

(c) an amino acid sequence of an extracellular domain of the polypeptideselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6),with its associated signal peptide sequence;

(d) an amino acid sequence of an extracellular domain of the polypeptideselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6),lacking its associated signal peptide sequence;

(e) an amino acid sequence encoded by the nucleotide sequence selectedfrom the group consisting of the nucleotide sequence shown in FIG. 1(SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or

(f) an amino acid sequence encoded by the full-length coding region ofthe nucleotide sequence selected from the group consisting of thenucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3)and FIG. 5 (SEQ ID NO: 5).

37. The oligopeptide of claim 35 or 36 which is conjugated to a growthinhibitory agent.

38. The oligopeptide of claim 35 or 36 which is conjugated to acytotoxic agent.

39. The oligopeptide of claim 38, wherein the cytotoxic agent isselected from the group consisting of toxins, antibiotics, radioactiveisotopes and nucleolytic enzymes.

40. The oligopeptide of claim 38, wherein the cytotoxic agent is atoxin.

41. The oligopeptide of claim 40, wherein the toxin is selected from thegroup consisting of maytansinoid and calicheamicin.

42. The oligopeptide of claim 40, wherein the toxin is a maytansinoid.

43. The oligopeptide of claim 35 or 36 which induces death of a cell towhich it binds.

44. The oligopeptide of claim 35 or 36 which is detectably labeled.

45. A TAHO binding organic molecule that binds to a polypeptide havingat least 80% amino acid sequence identity to:

(a) the polypeptide having the amino acid sequence selected from thegroup consisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6);

(b) the polypeptide having the amino acid sequence selected from thegroup consisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lacking itsassociated signal peptide;

(c) an extracellular domain of the polypeptide having the amino acidsequence selected from the group consisting of the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ IDNO: 6), with its associated signal peptide;

(d) an extracellular domain of the polypeptide having the amino acidsequence selected from the group consisting of the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ IDNO: 6), lacking its associated signal peptide;

(e) a polypeptide encoded by the nucleotide sequence selected from thegroup consisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or

(f) a polypeptide encoded by the full-length coding region of thenucleotide sequence selected from the group consisting of the nucleotidesequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5(SEQ ID NO: 5).

46. The organic molecule of claim 45 that binds to a polypeptide having:

(a) the amino acid sequence selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO:4) and FIG. 6 (SEQ ID NO: 6);

(b) the amino acid sequence selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO:4) and FIG. 6 (SEQ ID NO: 6), lacking its associated signal peptidesequence;

(c) an amino acid sequence of an extracellular domain of the polypeptideselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6),with its associated signal peptide sequence;

(d) an amino acid sequence of an extracellular domain of the polypeptideselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6),lacking its associated signal peptide sequence;

(e) an amino acid sequence encoded by the nucleotide sequence selectedfrom the group consisting of the nucleotide sequence shown in FIG. 1(SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or

(f) an amino acid sequence encoded by the full-length coding region ofthe nucleotide sequence selected from the group consisting of thenucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3)and FIG. 5 (SEQ ID NO: 5).

47. The organic molecule of claim 45 or 46 which is conjugated to agrowth inhibitory agent.

48. The organic molecule of claim 45 or 46 which is conjugated to acytotoxic agent.

49. The organic molecule of claim 48, wherein the cytotoxic agent isselected from the group consisting of toxins, antibiotics, radioactiveisotopes and nucleolytic enzymes.

50. The organic molecule of claim 48, wherein the cytotoxic agent is atoxin.

51. The organic molecule of claim 50, wherein the toxin is selected fromthe group consisting of maytansinoid and calicheamicin.

52. The organic molecule of claim 50, wherein the toxin is amaytansinoid.

53. The organic molecule of claim 45 or 46 which induces death of a cellto which it binds.

54. The organic molecule of claim 45 or 46 which is detectably labeled.

55. A composition of matter comprising:

(a) the polypeptide of claim 11;

(b) the polypeptide of claim 12;

(c) the antibody of claim 15;

(d) the antibody of claim 16;

(e) the oligopeptide of claim 35;

(f) the oligopeptide of claim 36;

(g) the TAHO binding organic molecule of claim 45; or

(h) the TAHO binding organic molecule of claim 46; in combination with acarrier.

56. The composition of matter of claim 55, wherein said carrier is apharmaceutically acceptable carrier.

57. An article of manufacture comprising:

(a) a container; and

(b) the composition of matter of claim 55 contained within saidcontainer.

58. The article of manufacture of claim 57 further comprising a labelaffixed to said container, or a package insert included with saidcontainer, referring to the use of said composition of matter for thetherapeutic treatment of or the diagnostic detection of a cancer.

59. A method of inhibiting the growth of a cell that expresses a proteinhaving at least 80% amino acid sequence identity to:

(a) the polypeptide having the amino acid sequence selected from thegroup consisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6);

(b) the polypeptide having the amino acid sequence selected from thegroup consisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lacking itsassociated signal peptide;

(c) an extracellular domain of the polypeptide having the amino acidsequence selected from the group consisting of the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ IDNO: 6), with its associated signal peptide;

(d) an extracellular domain of the polypeptide having the amino acidsequence selected from the group consisting of the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ IDNO: 6), lacking its associated signal peptide;

(e) a polypeptide encoded by the nucleotide sequence selected from thegroup consisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or

(f) a polypeptide encoded by the full-length coding region of thenucleotide sequence selected from the group consisting of the nucleotidesequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5(SEQ ID NO: 5), said method comprising contacting said cell with anantibody, oligopeptide or organic molecule that binds to said protein,the binding of said antibody, oligopeptide or organic molecule to saidprotein thereby causing an inhibition of growth of said cell.

60. The method of claim 59, wherein said antibody is a monoclonalantibody.

61. The method of claim 59, wherein said antibody is an antibodyfragment.

62. The method of claim 59, wherein said antibody is a chimeric or ahumanized antibody.

63. The method of claim 59, wherein said antibody, oligopeptide ororganic molecule is conjugated to a growth inhibitory agent.

64. The method of claim 59, wherein said antibody, oligopeptide ororganic molecule is conjugated to a cytotoxic agent.

65. The method of claim 64, wherein said cytotoxic agent is selectedfrom the group consisting of toxins, antibiotics, radioactive isotopesand nucleolytic enzymes.

66. The method of claim 64, wherein the cytotoxic agent is a toxin.

67. The method of claim 66, wherein the toxin is selected from the groupconsisting of maytansinoid and calicheamicin.

68. The method of claim 66, wherein the toxin is a maytansinoid.

69. The method of claim 59, wherein said antibody is produced inbacteria.

70. The method of claim 59, wherein said antibody is produced in CHOcells.

71. The method of claim 59, wherein said cell is a hematopoietic cell.

72. The method of claim 71, wherein said hematopoietic cell is selectedfrom the group consisting of a lymphocyte, leukocyte, platelet,erythrocyte and natural killer cell.

73. The method of claim 72, wherein said lymphocyte is a B cell or Tcell.

74. The method of claim 73 wherein said lymphocyte is a cancer cell.

75. The method of claim 74 wherein said cancer cell is further exposedto radiation treatment or a chemotherapeutic agent.

76. The method of claim 75, wherein said cancer cell is selected fromthe group consisting of a lymphoma cell, a myeloma cell and a leukemiacell.

77. The method of claim 71, wherein said protein is more abundantlyexpressed by said hematopoietic cell as compared to a non-hematopoieticcell.

78. The method of claim 59 which causes the death of said cell.

79. The method of claim 59, wherein said protein has:

(a) the amino acid sequence selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO:4) and FIG. 6 (SEQ ID NO: 6);

(b) the amino acid sequence selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO:4) and FIG. 6 (SEQ ID NO: 6), lacking its associated signal peptidesequence;

(c) an amino acid sequence of an extracellular domain of the polypeptideselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6),with its associated signal peptide sequence;

(d) an amino acid sequence of an extracellular domain of the polypeptideselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6),lacking its associated signal peptide sequence;

(e) an amino acid sequence encoded by the nucleotide sequence selectedfrom the group consisting of the nucleotide sequence shown in FIG. 1(SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or

(f) an amino acid sequence encoded by the full-length coding region ofthe nucleotide sequence selected from the group consisting of thenucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3)and FIG. 5 (SEQ ID NO: 5).

80. A method of therapeutically treating a mammal having a canceroustumor comprising cells that express a protein having at least 80% aminoacid sequence identity to:

(a) the polypeptide having the amino acid sequence selected from thegroup consisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6);

(b) the polypeptide having the amino acid sequence selected from thegroup consisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lacking itsassociated signal peptide;

(c) an extracellular domain of the polypeptide having the amino acidsequence selected from the group consisting of the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ IDNO: 6), with its associated signal peptide;

(d) an extracellular domain of the polypeptide having the amino acidsequence selected from the group consisting of the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ IDNO: 6), lacking its associated signal peptide;

(e) a polypeptide encoded by the nucleotide sequence selected from thegroup consisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or

(f) a polypeptide encoded by the full-length coding region of thenucleotide sequence selected from the group consisting of the nucleotidesequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5(SEQ ID NO: 5), said method comprising administering to said mammal atherapeutically effective amount of an antibody, oligopeptide or organicmolecule that binds to said protein, thereby effectively treating saidmammal.

81. The method of claim 80, wherein said antibody is a monoclonalantibody.

82. The method of claim 80, wherein said antibody is an antibodyfragment.

83. The method of claim 80, wherein said antibody is a chimeric or ahumanized antibody.

84. The method of claim 80, wherein said antibody, oligopeptide ororganic molecule is conjugated to a growth inhibitory agent.

85. The method of claim 80, wherein said antibody, oligopeptide ororganic molecule is conjugated to a cytotoxic agent.

86. The method of claim 85, wherein said cytotoxic agent is selectedfrom the group consisting of toxins, antibiotics, radioactive isotopesand nucleolytic enzymes.

87. The method of claim 85, wherein the cytotoxic agent is a toxin.

88. The method of claim 87, wherein the toxin is selected from the groupconsisting of maytansinoid and calicheamicin.

89. The method of claim 87, wherein the toxin is a maytansinoid.

90. The method of claim 80, wherein said antibody is produced inbacteria.

91. The method of claim 80, wherein said antibody is produced in CHOcells.

92. The method of claim 80, wherein said tumor is further exposed toradiation treatment or a chemotherapeutic agent.

93. The method of claim 80, wherein said tumor is a lymphoma, leukemiaor myeloma tumor.

94. The method of claim 80, wherein said protein is more abundantlyexpressed by a hematopoietic cell as compared to a non-hematopoieticcell of said tumor.

95. The method of claim 94, wherein said protein is more abundantlyexpressed by cancerous hematopoietic cells of said tumor as compared tonormal hematopoietic cells of said tumor.

96. The method of claim 80, wherein said protein has:

(a) the amino acid sequence selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO:4) and FIG. 6 (SEQ ID NO: 6);

(b) the amino acid sequence selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO:4) and FIG. 6 (SEQ ID NO: 6), lacking its associated signal peptidesequence;

(c) an amino acid sequence of an extracellular domain of the polypeptideselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6),with its associated signal peptide sequence;

(d) an amino acid sequence of an extracellular domain of the polypeptideselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6),lacking its associated signal peptide sequence;

(e) an amino acid sequence encoded by the nucleotide sequence selectedfrom the group consisting of the nucleotide sequence shown in FIG. 1(SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or

(f) an amino acid sequence encoded by the full-length coding region ofthe nucleotide sequence selected from the group consisting of thenucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3)and FIG. 5 (SEQ ID NO: 5).

97. A method of determining the presence of a protein in a samplesuspected of containing said protein, wherein said protein has at least80% amino acid sequence identity to:

(a) the polypeptide having the amino acid sequence selected from thegroup consisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6);

(b) the polypeptide having the amino acid sequence selected from thegroup consisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lacking itsassociated signal peptide;

(c) an extracellular domain of the polypeptide having the amino acidsequence selected from the group consisting of the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ IDNO: 6), with its associated signal peptide;

(d) an extracellular domain of the polypeptide having the amino acidsequence selected from the group consisting of the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ IDNO: 6), lacking its associated signal peptide;

(e) a polypeptide encoded by the nucleotide sequence selected from thegroup consisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or

(f) a polypeptide encoded by the full-length coding region of thenucleotide sequence selected from the group consisting of the nucleotidesequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5(SEQ ID NO: 5), said method comprising exposing said sample to anantibody, oligopeptide or organic molecule that binds to said proteinand determining binding of said antibody, oligopeptide or organicmolecule to said protein in said sample, wherein binding of theantibody, oligopeptide or organic molecule to said protein is indicativeof the presence of said protein in said sample.

98. The method of claim 97, wherein said sample comprises a cellsuspected of expressing said protein.

99. The method of claim 98, wherein said cell is a cancer cell.

100. The method of claim 97, wherein said antibody, oligopeptide ororganic molecule is detectably labeled.

101. The method of claim 97, wherein said protein has:

(a) the amino acid sequence selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO:4) and FIG. 6 (SEQ ID NO: 6);

(b) the amino acid sequence selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO:4) and FIG. 6 (SEQ ID NO: 6), lacking its associated signal peptidesequence;

(c) an amino acid sequence of an extracellular domain of the polypeptideselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6),with its associated signal peptide sequence;

(d) an amino acid sequence of an extracellular domain of the polypeptideselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6),lacking its associated signal peptide sequence;

(e) an amino acid sequence encoded by the nucleotide sequence selectedfrom the group consisting of the nucleotide sequence shown in FIG. 1(SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or

(f) an amino acid sequence encoded by the full-length coding region ofthe nucleotide sequence selected from the group consisting of thenucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3)and FIG. 5 (SEQ ID NO: 5).

102. A method for treating or preventing a cell proliferative disorderassociated with increased expression or activity of a protein having atleast 80% amino acid sequence identity to:

(a) the polypeptide having the amino acid sequence selected from thegroup consisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6);

(b) the polypeptide having the amino acid sequence selected from thegroup consisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lacking itsassociated signal peptide;

(c) an extracellular domain of the polypeptide having the amino acidsequence selected from the group consisting of the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ IDNO: 6) with its associated signal peptide;

(d) an extracellular domain of the polypeptide having the amino acidsequence selected from the group consisting of the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ IDNO: 6), lacking its associated signal peptide;

(e) a polypeptide encoded by the nucleotide sequence selected from thegroup consisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or

(f) a polypeptide encoded by the full-length coding region of thenucleotide sequence selected from the group consisting of the nucleotidesequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5(SEQ ID NO: 5), said method comprising administering to a subject inneed of such treatment an effective amount of an antagonist of saidprotein, thereby effectively treating or preventing said cellproliferative disorder.

103. The method of claim 102, wherein said cell proliferative disorderis cancer.

104. The method of claim 102, wherein said antagonist is an anti-TAHOpolypeptide antibody, TAHO binding oligopeptide, TAHO binding organicmolecule or antisense oligonucleotide.

105. A method of binding an antibody, oligopeptide or organic moleculeto a cell that expresses a protein having at least 80% amino acidsequence identity to:

(a) the polypeptide having the amino acid sequence selected from thegroup consisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6);

(b) the polypeptide having the amino acid sequence selected from thegroup consisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lacking itsassociated signal peptide;

(c) an extracellular domain of the polypeptide having the amino acidsequence selected from the group consisting of the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ IDNO: 6), with its associated signal peptide;

(d) an extracellular domain of the polypeptide having the amino acidsequence selected from the group consisting of the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ IDNO: 6), lacking its associated signal peptide;

(e) a polypeptide encoded by the nucleotide sequence selected from thegroup consisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or

(f) a polypeptide encoded by the full-length coding region of thenucleotide sequence selected from the group consisting of the nucleotidesequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5(SEQ ID NO: 5), said method comprising contacting said cell with anantibody, oligopeptide or organic molecule that binds to said proteinand allowing the binding of the antibody, oligopeptide or organicmolecule to said protein to occur, thereby binding said antibody,oligopeptide or organic molecule to said cell.

106. The method of claim 105, wherein said antibody is a monoclonalantibody.

107. The method of claim 105, wherein said antibody is an antibodyfragment.

108. The method of claim 105, wherein said antibody is a chimeric or ahumanized antibody.

109. The method of claim 105, wherein said antibody, oligopeptide ororganic molecule is conjugated to a growth inhibitory agent.

110. The method of claim 105, wherein said antibody, oligopeptide ororganic molecule is conjugated to a cytotoxic agent.

111. The method of claim 110, wherein said cytotoxic agent is selectedfrom the group consisting of toxins, antibiotics, radioactive isotopesand nucleolytic enzymes.

112. The method of claim 110, wherein the cytotoxic agent is a toxin.

113. The method of claim 112, wherein the toxin is selected from thegroup consisting of maytansinoid and calicheamicin.

114. The method of claim 112, wherein the toxin is a maytansinoid.

115. The method of claim 105, wherein said antibody is produced inbacteria.

116. The method of claim 105, wherein said antibody is produced in CHOcells.

117. The method of claim 105, wherein said cell is a hematopoietic cell.

118. The method of claim 117, wherein said hematopoietic cell is aselected from the group consisting of a lymphocyte, leukocyte, platelet,erythrocyte and natural killer cell.

119. The method of claim 118, wherein said lymphocyte is a B cell or a Tcell.

120. The method of claim 119, wherein said lymphocyte is a cancer cell.

121. The method of claim 120 wherein said cancer cell is further exposedto radiation treatment or a chemotherapeutic agent.

122. The method of claim 120, wherein said cancer cell is selected fromthe group consisting of a leukemia cell, a lymphoma cell and a myelomacell.

123. The method of claim 120, wherein said protein is more abundantlyexpressed by said hematopoietic cell as compared to a non-hematopoieticcell.

124. The method of claim 105 which causes the death of said cell.

125. Use of a nucleic acid as claimed in any of claims 1 to 5 or 30 inthe preparation of a medicament for the therapeutic treatment ordiagnostic detection of a cancer.

126. Use of a nucleic acid as claimed in any of claims 1 to 5 or 30 inthe preparation of a medicament for treating a tumor.

127. Use of a nucleic acid as claimed in any of claims 1 to 5 in thepreparation of a medicament for treatment or prevention of a cellproliferative disorder.

128. Use of an expression vector as claimed in claim 6 in thepreparation of a medicament for the therapeutic treatment or diagnosticdetection of a cancer.

129. Use of an expression vector as claimed in claim 6 in thepreparation of medicament for treating a tumor.

130. Use of an expression vector as claimed in claim 6 in thepreparation of a medicament for treatment or prevention of a cellproliferative disorder.

131. Use of a host cell as claimed in claim 8 in the preparation of amedicament for the therapeutic treatment or diagnostic detection of acancer.

132. Use of a host cell as claimed in claim 8 in the preparation of amedicament for treating a tumor.

133. Use of a host cell as claimed in claim 8 in the preparation of amedicament for treatment or prevention of a cell proliferative disorder.

134. Use of a polypeptide as claimed in claim 11 or 12 in thepreparation of a medicament for the therapeutic treatment or diagnosticdetection of a cancer.

135. Use of a polypeptide as claimed in claim 11 or 12 in thepreparation of a medicament for treating a tumor.

136. Use of a polypeptide as claimed in claim 11 or 12 in thepreparation of a medicament for treatment or prevention of a cellproliferative disorder.

137. Use of an antibody as claimed in claim 15 or 16 in the preparationof a medicament for the therapeutic treatment or diagnostic detection ofa cancer.

138. Use of an antibody as claimed in claim 15 or 16 in the preparationof a medicament for treating a tumor.

139. Use of an antibody as claimed in claim 15 or 16 in the preparationof a medicament for treatment or prevention of a cell proliferativedisorder.

140. Use of an oligopeptide as claimed in claim 35 or 36 in thepreparation of a medicament for the therapeutic treatment or diagnosticdetection of a cancer.

141. Use of an oligopeptide as claimed in claim 35 or 36 in thepreparation of a medicament for treating a tumor.

142. Use of an oligopeptide as claimed in claim 35 or 36 in thepreparation of a medicament for treatment or prevention of a cellproliferative disorder.

143. Use of a TAHO binding organic molecule as claimed in claim 45 or 46in the preparation of a medicament for the therapeutic treatment ordiagnostic detection of a cancer.

144. Use of a TAHO binding organic molecule as claimed in claim 45 or 46in the preparation of a medicament for treating a tumor.

145. Use of a TAHO binding organic molecule as claimed in claims 45 or46 in the preparation of a medicament for treatment or prevention of acell proliferative disorder.

146. Use of a composition of matter as claimed in claim 55 in thepreparation of a medicament for the therapeutic treatment or diagnosticdetection of a cancer.

147. Use of a composition of matter as claimed in claim 55 in thepreparation of a medicament for treating a tumor.

148. Use of a composition of matter as claimed in claim 55 in thepreparation of a medicament for treatment or prevention of a cellproliferative disorder.

149. Use of an article of manufacture as claimed in claim 57 in thepreparation of a medicament for the therapeutic treatment or diagnosticdetection of a cancer.

150. Use of an article of manufacture as claimed in claim 58 in thepreparation of a medicament for treating a tumor.

151. Use of an article of manufacture as claimed in claim 58 in thepreparation of a medicament for treatment or prevention of a cellproliferative disorder.

152. A method for inhibiting the growth of a cell, wherein the growth ofsaid cell is at least in part dependent upon a growth potentiatingeffect of a protein having at least 80% amino acid sequence identity to:

(a) the polypeptide having the amino acid sequence selected from thegroup consisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6);

(b) the polypeptide having the amino acid sequence selected from thegroup consisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lacking itsassociated signal peptide;

(c) an extracellular domain of the polypeptide having the amino acidsequence selected from the group consisting of the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ IDNO: 6), with its associated signal peptide;

(d) an extracellular domain of the polypeptide having the amino acidsequence selected from the group consisting of the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ IDNO: 6), lacking its associated signal peptide;

(e) a polypeptide encoded by the nucleotide sequence selected from thegroup consisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or

(f) a polypeptide encoded by the full-length coding region of thenucleotide sequence selected from the group consisting of the nucleotidesequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5(SEQ ID NO: 5), said method comprising contacting said protein with anantibody, oligopeptide or organic molecule that binds to said protein,there by inhibiting the growth of said cell.

153. The method of claim 152, wherein said cell is a hematopoietic cell.

154. The method of claim 152, wherein said protein is expressed by saidcell.

155. The method of claim 152, wherein the binding of said antibody,oligopeptide or organic molecule to said protein antagonizes a cellgrowth-potentiating activity of said protein.

156. The method of claim 152, wherein the binding of said antibody,oligopeptide or organic molecule to said protein induces the death ofsaid cell.

157. The method of claim 152, wherein said antibody is a monoclonalantibody.

158. The method of claim 152, wherein said antibody is an antibodyfragment.

159. The method of claim 152, wherein said antibody is a chimeric or ahumanized antibody.

160. The method of claim 152, wherein said antibody, oligopeptide ororganic molecule is conjugated to a growth inhibitory agent.

161. The method of claim 152, wherein said antibody, oligopeptide ororganic molecule is conjugated to a cytotoxic agent.

162. The method of claim 161, wherein said cytotoxic agent is selectedfrom the group consisting of toxins, antibiotics, radioactive isotopesand nucleolytic enzymes.

163. The method of claim 161, wherein the cytotoxic agent is a toxin.

164. The method of claim 163, wherein the toxin is selected from thegroup consisting of maytansinoid and calicheamicin.

165. The method of claim 163, wherein the toxin is a maytansinoid.

166. The method of claim 152, wherein said antibody is produced inbacteria.

167. The method of claim 152, wherein said antibody is produced in CHOcells.

168. The method of claim 152, wherein said protein has:

(a) the amino acid sequence selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO:4) and FIG. 6 (SEQ ID NO: 6);

(b) the amino acid sequence selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO:4) and FIG. 6 (SEQ ID NO: 6), lacking its associated signal peptidesequence;

(c) an amino acid sequence of an extracellular domain of the polypeptideselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6),with its associated signal peptide sequence;

(d) an amino acid sequence of an extracellular domain of the polypeptideselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6),lacking its associated signal peptide sequence;

(e) an amino acid sequence encoded by the nucleotide sequence selectedfrom the group consisting of the nucleotide sequence shown in FIG. 1(SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or

(f) an amino acid sequence encoded by the full-length coding region ofthe nucleotide sequence selected from the group consisting of thenucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3)and FIG. 5 (SEQ ID NO: 5).

169. A method of therapeutically treating a tumor in a mammal, whereinthe growth of said tumor is at least in part dependent upon a growthpotentiating effect of a protein having at least 80% amino acid sequenceidentity to:

(a) the polypeptide having the amino acid sequence selected from thegroup consisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6);

(b) the polypeptide having the amino acid sequence selected from thegroup consisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lacking itsassociated signal peptide;

(c) an extracellular domain of the polypeptide having the amino acidsequence selected from the group consisting of the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ IDNO: 6), with its associated signal peptide;

(d) an extracellular domain of the polypeptide having the amino acidsequence selected from the group consisting of the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ IDNO: 6), lacking its associated signal peptide;

(e) a polypeptide encoded by the nucleotide sequence selected from thegroup consisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or

(f) a polypeptide encoded by the full-length coding region of thenucleotide sequence selected from the group consisting of the nucleotidesequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5(SEQ ID NO: 5), said method comprising contacting said protein with anantibody, oligopeptide or organic molecule that binds to said protein,thereby effectively treating said tumor.

170. The method of claim 169, wherein said protein is expressed by cellsof said tumor.

171. The method of claim 169, wherein the binding of said antibody,oligopeptide or organic molecule to said protein antagonizes a cellgrowth-potentiating activity of said protein.

172. The method of claim 169, wherein said antibody is a monoclonalantibody.

173. The method of claim 169, wherein said antibody is an antibodyfragment.

174. The method of claim 169, wherein said antibody is a chimeric or ahumanized antibody.

175. The method of claim 169, wherein said antibody, oligopeptide ororganic molecule is conjugated to a growth inhibitory agent.

176. The method of claim 169, wherein said antibody, oligopeptide ororganic molecule is conjugated to a cytotoxic agent.

177. The method of claim 176, wherein said cytotoxic agent is selectedfrom the group consisting of toxins, antibiotics, radioactive isotopesand nucleolytic enzymes.

178. The method of claim 176, wherein the cytotoxic agent is a toxin.

179. The method of claim 178, wherein the toxin is selected from thegroup consisting of maytansinoid and calicheamicin.

180. The method of claim 178, wherein the toxin is a maytansinoid.

181. The method of claim 169, wherein said antibody is produced inbacteria.

182. The method of claim 169, wherein said antibody is produced in CHOcells.

183. The method of claim 169, wherein said protein has:

(a) the amino acid sequence selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO:4) and FIG. 6 (SEQ ID NO: 6);

(b) the amino acid sequence selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO:4) and FIG. 6 (SEQ ID NO: 6), lacking its associated signal peptidesequence;

(c) an amino acid sequence of an extracellular domain of the polypeptideselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6),with its associated signal peptide sequence;

(d) an amino acid sequence of an extracellular domain of the polypeptideselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6),lacking its associated signal peptide sequence;

(e) an amino acid sequence encoded by the nucleotide sequence selectedfrom the group consisting of the nucleotide sequence shown in FIG. 1(SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or

(f) an amino acid sequence encoded by the full-length coding region ofthe nucleotide sequence selected from the group consisting of thenucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3)and FIG. 5 (SEQ ID NO: 5).

184. A composition of matter comprising the chimeric polypeptide ofclaim 13.

185. Use of a nucleic acid as claimed in claim 30 in the preparation ofa medicament for treatment or prevention of a cell proliferativedisorder.

186. Use of an expression vector as claimed in claim 7 in thepreparation of a medicament for the therapeutic treatment or diagnosticdetection of a cancer.

187. Use of an expression vector as claimed in claim 31 in thepreparation of a medicament for the therapeutic treatment or diagnosticdetection of a cancer.

188. Use of an expression vector as claimed in claim 7 in thepreparation of medicament for treating a tumor.

189. Use of an expression vector as claimed in claim 31 in thepreparation of medicament for treating a tumor.

190. Use of an expression vector as claimed in claim 7 in thepreparation of a medicament for treatment or prevention of a cellproliferative disorder.

191. Use of an expression vector as claimed in claim 31 in thepreparation of a medicament for treatment or prevention of a cellproliferative disorder.

192. Use of a host cell as claimed in claim 9 in the preparation of amedicament for the therapeutic treatment or diagnostic detection of acancer.

193. Use of a host cell as claimed in claim 32 in the preparation of amedicament for the therapeutic treatment or diagnostic detection of acancer.

194. Use of a host cell as claimed in claim 33 in the preparation of amedicament for the therapeutic treatment or diagnostic detection of acancer.

195. Use of a host cell as claimed in claim 9 in the preparation of amedicament for treating a tumor.

196. Use of a host cell as claimed in claim 32 in the preparation of amedicament for treating a tumor.

197. Use of a host cell as claimed in claim 33 in the preparation of amedicament for treating a tumor.

198. Use of a host cell as claimed in claim 9 in the preparation of amedicament for treatment or prevention of a cell proliferative disorder.

199. Use of a host cell as claimed in claim 32 in the preparation of amedicament for treatment or prevention of a cell proliferative disorder.

200. Use of a host cell as claimed in claim 33 in the preparation of amedicament for treatment or prevention of a cell proliferative disorder.

201. Use of a polypeptide as claimed in claim 13 in the preparation of amedicament for the therapeutic treatment or diagnostic detection of acancer.

202. Use of a polypeptide as claimed in claim 14 in the preparation of amedicament for the therapeutic treatment or diagnostic detection of acancer.

203. Use of a polypeptide as claimed in claim 13 in the preparation of amedicament for treating a tumor.

204. Use of a polypeptide as claimed in claim 14 in the preparation of amedicament for treating at tumor.

205. Use of a polypeptide as claimed in claim 13 in the preparation of amedicament for treatment or prevention of a cell proliferative disorder.

206. Use of a polypeptide as claimed in claim 14 in the preparation of amedicament for treatment or prevention of a cell proliferative disorder.

207. Use of an antibody as claimed in claim 17 in the preparation of amedicament for the therapeutic treatment or diagnostic detection of acancer.

208. Use of an antibody as claimed in claim 18 in the preparation of amedicament for the therapeutic treatment or diagnostic detection of acancer.

209. Use of an antibody as claimed in claim 19 in the preparation of amedicament for the therapeutic treatment or diagnostic detection of acancer.

210. Use of an antibody as claimed in claim 20 in the preparation of amedicament for the therapeutic treatment or diagnostic detection of acancer.

211. Use of an antibody as claimed in claim 21 in the preparation of amedicament for the therapeutic treatment or diagnostic detection of acancer.

212. Use of an antibody as claimed in claim 22 in the preparation of amedicament for the therapeutic treatment or diagnostic detection of acancer.

213. Use of an antibody as claimed in claim 23 in the preparation of amedicament for the therapeutic treatment or diagnostic detection of acancer.

214. Use of an antibody as claimed in claim 24 in the preparation of amedicament for the therapeutic treatment or diagnostic detection of acancer.

215. Use of an antibody as claimed in claim 25 in the preparation of amedicament for the therapeutic treatment or diagnostic detection of acancer.

216. Use of an antibody as claimed in claim 26 in the preparation of amedicament for the therapeutic treatment or diagnostic detection of acancer.

217. Use of an antibody as claimed in claim 27 in the preparation of amedicament for the therapeutic treatment or diagnostic detection of acancer.

218. Use of an antibody as claimed in claim 28 in the preparation of amedicament for the therapeutic treatment or diagnostic detection of acancer.

219. Use of an antibody as claimed in claim 29 in the preparation of amedicament for the therapeutic treatment or diagnostic detection of acancer.

220. Use of an antibody as claimed in claim 17 in the preparation of amedicament for treating a tumor.

221. Use of an antibody as claimed in claim 18 in the preparation of amedicament for treating a tumor.

222. Use of an antibody as claimed in claim 19 in the preparation of amedicament for treating a tumor.

223. Use of an antibody as claimed in claim 20 in the preparation of amedicament for treating a tumor.

224. Use of an antibody as claimed in claim 21 in the preparation of amedicament for treating a tumor.

225. Use of an antibody as claimed in claim 22 in the preparation of amedicament for treating a tumor.

226. Use of an antibody as claimed in claim 23 in the preparation of amedicament for treating a tumor.

227. Use of an antibody as claimed in claim 24 in the preparation of amedicament for treating a tumor.

228. Use of an antibody as claimed in claim 25 in the preparation of amedicament for treating a tumor.

229. Use of an antibody as claimed in claim 26 in the preparation of amedicament for treating a tumor.

230. Use of an antibody as claimed in claim 27 in the preparation of amedicament for treating a tumor.

231. Use of an antibody as claimed in claim 28 in the preparation of amedicament for treating a tumor.

232. Use of an antibody as claimed in claim 29 in the preparation of amedicament for treating a tumor.

233. Use of an antibody as claimed in claim 17 in the preparation of amedicament for treatment or prevention of a cell proliferative disorder.

234. Use of an antibody as claimed in claim 18 in the preparation of amedicament for treatment or prevention of a cell proliferative disorder.

235. Use of an antibody as claimed in claim 17 in the preparation of amedicament for treatment or prevention of a cell proliferative disorder.

235. Use of an antibody as claimed in claim 18 in the preparation of amedicament for treatment or prevention of a cell proliferative disorder.

237. Use of an antibody as claimed in claim 19 in the preparation of amedicament for treatment or prevention of a cell proliferative disorder.

238. Use of an antibody as claimed in claim 20 in the preparation of amedicament for treatment or prevention of a cell proliferative disorder.

239. Use of an antibody as claimed in claim 21 in the preparation of amedicament for treatment or prevention of a cell proliferative disorder.

240. Use of an antibody as claimed in claim 22 in the preparation of amedicament for treatment or prevention of a cell proliferative disorder.

241. Use of an antibody as claimed in claim 23 in the preparation of amedicament for treatment or prevention of a cell proliferative disorder.

242. Use of an antibody as claimed in claim 24 in the preparation of amedicament for treatment or prevention of a cell proliferative disorder.

243. Use of an antibody as claimed in claim 25 in the preparation of amedicament for treatment or prevention of a cell proliferative disorder.

244. Use of an antibody as claimed in claim 26 in the preparation of amedicament for treatment or prevention of a cell proliferative disorder.

245. Use of an antibody as claimed in claim 27 in the preparation of amedicament for treatment or prevention of a cell proliferative disorder.

246. Use of an antibody as claimed in claim 28 in the preparation of amedicament for treatment or prevention of a cell proliferative disorder.

247. Use of an antibody as claimed in claim 29 in the preparation of amedicament for treatment or prevention of a cell proliferative disorder.

248. Use of an oligopeptide as claimed in claim 37 in the preparation ofa medicament for the therapeutic treatment or diagnostic detection of acancer.

249. Use of an oligopeptide as claimed in claim 38 in the preparation ofa medicament for the therapeutic treatment or diagnostic detection of acancer.

250. Use of an oligopeptide as claimed in claim 39 in the preparation ofa medicament for the therapeutic treatment or diagnostic detection of acancer.

251. Use of an oligopeptide as claimed in claim 40 in the preparation ofa medicament for the therapeutic treatment or diagnostic detection of acancer.

252. Use of an oligopeptide as claimed in claim 41 in the preparation ofa medicament for the therapeutic treatment or diagnostic detection of acancer.

253. Use of an oligopeptide as claimed in claim 42 in the preparation ofa medicament for the therapeutic treatment or diagnostic detection of acancer.

254. Use of an oligopeptide as claimed in claim 43 in the preparation ofa medicament for the therapeutic treatment or diagnostic detection of acancer.

255. Use of an oligopeptide as claimed in claim 44 in the preparation ofa medicament for the therapeutic treatment or diagnostic detection of acancer.

256. Use of an oligopeptide as claimed in claim 37 in the preparation ofa medicament for treating a tumor.

257. Use of an oligopeptide as claimed in claim 38 in the preparation ofa medicament for treating a tumor.

258. Use of an oligopeptide as claimed in claim 39 in the preparation ofa medicament for treating a tumor.

259. Use of an oligopeptide as claimed in claim 40 in the preparation ofa medicament for treating a tumor.

260. Use of an oligopeptide as claimed in claim 41 in the preparation ofa medicament for treating a tumor.

261. Use of an oligopeptide as claimed in claim 42 in the preparation ofa medicament for treating a tumor.

262. Use of an oligopeptide as claimed in claim 43 in the preparation ofa medicament for treating a tumor.

263. Use of an oligopeptide as claimed in claim 44 in the preparation ofa medicament for treating a tumor.

264. Use of an oligopeptide as claimed in claim 37 in the preparation ofa medicament for treatment or prevention of a cell proliferativedisorder.

265. Use of an oligopeptide as claimed in claim 38 in the preparation ofa medicament for treatment or prevention of a cell proliferativedisorder.

266. Use of an oligopeptide as claimed in claim 39 in the preparation ofa medicament for treatment or prevention of a cell proliferativedisorder.

267. Use of an oligopeptide as claimed in claim 40 in the preparation ofa medicament for treatment or prevention of a cell proliferativedisorder.

268. Use of an oligopeptide as claimed in claim 41 in the preparation ofa medicament for treatment or prevention of a cell proliferativedisorder.

269. Use of an oligopeptide as claimed in claim 42 in the preparation ofa medicament for treatment or prevention of a cell proliferativedisorder.

270. Use of an oligopeptide as claimed in claim 43 in the preparation ofa medicament for treatment or prevention of a cell proliferativedisorder.

271. Use of an oligopeptide as claimed in claim 44 in the preparation ofa medicament for treatment or prevention of a cell proliferativedisorder.

272. Use of a TAHO binding organic molecule as claimed in claim 47 inthe preparation of a medicament for the therapeutic treatment ordiagnostic detection of a cancer.

273. Use of a TAHO binding organic molecule as claimed in claim 48 inthe preparation of a medicament for the therapeutic treatment ordiagnostic detection of a cancer.

274. Use of a TAHO binding organic molecule as claimed in claim 49 inthe preparation of a medicament for the therapeutic treatment ordiagnostic detection of a cancer.

275. Use of a TAHO binding organic molecule as claimed in claim 50 inthe preparation of a medicament for the therapeutic treatment ordiagnostic detection of a cancer.

276. Use of a TAHO binding organic molecule as claimed in claim 51 inthe preparation of a medicament for the therapeutic treatment ordiagnostic detection of a cancer.

277. Use of a TAHO binding organic molecule as claimed in claim 52 inthe preparation of a medicament for the therapeutic treatment ordiagnostic detection of a cancer.

278. Use of a TAHO binding organic molecule as claimed in claim 53 inthe preparation of a medicament for the therapeutic treatment ordiagnostic detection of a cancer.

279. Use of a TAHO binding organic molecule as claimed in claim 54 inthe preparation of a medicament for the therapeutic treatment ordiagnostic detection of a cancer.

280. Use of a TAHO binding organic molecule as claimed in claim 47 inthe preparation of a medicament for treating a tumor.

281. Use of a TAHO binding organic molecule as claimed in claim 48 inthe preparation of a medicament for treating a tumor.

282. Use of a TAHO binding organic molecule as claimed in claim 49 inthe preparation of a medicament for treating a tumor.

283. Use of a TAHO binding organic molecule as claimed in claim 50 inthe preparation of a medicament for treating a tumor.

284. Use of a TAHO binding organic molecule as claimed in claim 51 inthe preparation of a medicament for treating a tumor.

285. Use of a TAHO binding organic molecule as claimed in claim 52 inthe preparation of a medicament for treating a tumor.

286. Use of a TAHO binding organic molecule as claimed in claim 53 inthe preparation of a medicament for treating a tumor.

287. Use of a TAHO binding organic molecule as claimed in claim 54 inthe preparation of a medicament for treating a tumor.

288. Use of a TAHO binding organic molecule as claimed in claim 47 inthe preparation of a medicament for treatment or prevention of a cellproliferative disorder.

289. Use of a TAHO binding organic molecule as claimed in claim 48 inthe preparation of a medicament for treatment or prevention of a cellproliferative disorder.

290. Use of a TAHO binding organic molecule as claimed in claim 49 inthe preparation of a medicament for treatment or prevention of a cellproliferative disorder.

291. Use of a TAHO binding organic molecule as claimed in claim 50 inthe preparation of a medicament for treatment or prevention of a cellproliferative disorder.

292. Use of a TAHO binding organic molecule as claimed in claim 51 inthe preparation of a medicament for treatment or prevention of a cellproliferative disorder.

293. Use of a TAHO binding organic molecule as claimed in claim 52 inthe preparation of a medicament for treatment or prevention of a cellproliferative disorder.

294. Use of a TAHO binding organic molecule as claimed in claim 53 inthe preparation of a medicament for treatment or prevention of a cellproliferative disorder.

295. Use of a TAHO binding organic molecule as claimed in claim 54 inthe preparation of a medicament for treatment or prevention of a cellproliferative disorder.

296. Use of a composition of matter as claimed in claim 56 in thepreparation of a medicament for the therapeutic treatment or diagnosticdetection of a cancer.

297. Use of a composition of matter as claimed in claim 56 in thepreparation of a medicament for treating a tumor.

298. Use of a composition of matter as claimed in claim 56 in thepreparation of a medicament for treatment or prevention of a cellproliferative disorder.

299. Use of an article of manufacture as claimed in claim 58 in thepreparation of a medicament for the therapeutic treatment or diagnosticdetection of a cancer.

300. Use of an article of manufacture as claimed in claim 58 in thepreparation of a medicament for treating a tumor.

301. Use of an article of manufacture as claimed in claim 58 in thepreparation of a medicament for treatment or prevention of a cellproliferative disorder.

302. A method of patient selection for treatment of tumors, said methodcomprising detecting in tumor samples from prospective patients cellsthat have:

(a) low or no expression of a polypeptide having an amino acid sequencehaving at least 95% amino acid sequence identity to the polypeptidehaving the amino acid sequence selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2) and FIG. 6 (SEQ IDNO: 6) and significant expression of a second polypeptide having anamino acid sequence having at least 95% amino acid sequence identity tothe polypeptide having the amino acid sequence shown in FIG. 4 (SEQ IDNO: 4);

(b) low or no expression of a polypeptide having an amino acid sequencehaving at least 95% amino acid sequence identity to the polypeptidehaving the amino acid sequence selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2) and FIG. 6 (SEQ IDNO: 6), lacking its associated signal peptide, and significantexpression of a second polypeptide having an amino acid sequence havingat least 95% amino acid sequence identity to the polypeptide having theamino acid sequence shown in FIG. 4 (SEQ ID NO: 4), lacking itsassociated signal peptide;

(c) low or no expression of a polypeptide having an amino acid sequencehaving at least 95% amino acid sequence identity to the polypeptidehaving the amino acid sequence selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2) and FIG. 6 (SEQ IDNO: 6), with its associated signal peptide, and significant expressionof a second polypeptide having an amino acid sequence having at least95% amino acid sequence identity to the polypeptide having the aminoacid sequence shown in FIG. 4 (SEQ ID NO: 4), with its associated signalpeptide;

(d) low or no expression of a polypeptide having an amino acid sequencehaving at least 95% amino acid sequence identity to an extracellulardomain of the polypeptide having the amino acid sequence selected fromthe group consisting of the amino acid sequence shown in FIG. 2 (SEQ IDNO: 2) and FIG. 6 (SEQ ID NO: 6), lacking its associated signal peptide,and significant expression of a second polypeptide having an amino acidsequence having at least 95% amino acid sequence identity to anextracellular domain of the polypeptide having the amino acid sequenceshown in FIG. 4 (SEQ ID NO: 4), lacking its associated signal peptide;

(e) low or no expression of a polypeptide having an amino acid sequencehaving at least 95% amino acid sequence identity to a polypeptideencoded by the nucleotide sequence selected from the group consisting ofthe nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1) and FIG. 5 (SEQID NO: 5) and significant expression of a second polypeptide having anamino acid sequence having at least 95% amino acid sequence identity toa polypeptide encoded by the nucleotide sequence shown in FIG. 3 (SEQ IDNO: 3); or

(f) low or no expression of a polypeptide having an amino acid sequencehaving at least 95% amino acid sequence identity to a polypeptideencoded by the full-length coding region of the nucleotide sequenceselected from the group consisting of the nucleotide sequence shown inFIG. 1 (SEQ ID NO: 1) and FIG. 5 (SEQ ID NO: 5) and significantexpression of a second polypeptide having an amino acid sequence havingat least 95% amino acid sequence identity to a polypeptide encoded bythe full-length coding region of the nucleotide sequence shown in FIG. 3(SEQ ID NO: 3),

wherein the identification of said cells from said tumor samplesindicates the identification of a patient for treatment with anantibody, oligopeptide or organic molecule, conjugated to agrowth-inhibitory agent or a cytotoxic agent, said conjugated antibody,oligopeptide or organic molecule binds to said second polypeptide anddepends on internalization into said cells for effective treatment.

303. A method of patient selection for treatment of tumors, said methodcomprising detecting in tumor samples from prospective patients cellsthat have:

(a) significant expression of a polypeptide having an amino acidsequence having at least 95% amino acid sequence identity to thepolypeptide having the amino acid sequence selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2) andFIG. 6 (SEQ ID NO: 6) in a minority of the cells or in a population ofcells distinct from cells that with significant expression of a secondpolypeptide having an amino acid sequence having at least 95% amino acidsequence identity to the polypeptide having the amino acid sequenceshown in FIG. 4 (SEQ ID NO: 4);

(b) significant expression of a polypeptide having an amino acidsequence having at least 95% amino acid sequence identity to thepolypeptide having the amino acid sequence selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2) andFIG. 6 (SEQ ID NO: 6), lacking its associated signal peptide, in aminority of the cells or in a population of cells distinct from cellswith significant expression of a second polypeptide having an amino acidsequence having at least 95% amino acid sequence identity to thepolypeptide having the amino acid sequence shown in FIG. 4 (SEQ ID NO:4), lacking its associated signal peptide;

(c) significant expression of a polypeptide having an amino acidsequence having at least 95% amino acid sequence identity to thepolypeptide having the amino acid sequence selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2) andFIG. 6 (SEQ ID NO: 6), with its associated signal peptide, in a minorityof the cells or in a population of cells distinct from cells withsignificant expression of a second polypeptide having an amino acidsequence having at least 95% amino acid sequence identity to thepolypeptide having the amino acid sequence shown in FIG. 4 (SEQ ID NO:4), with its associated signal peptide;

(d) significant expression of a polypeptide having an amino acidsequence having at least 95% amino acid sequence identity to anextracellular domain of the polypeptide having the amino acid sequenceselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2) and FIG. 6 (SEQ ID NO: 6), lacking its associatedsignal peptide, in a minority of the cells or in a population of cellsdistinct from cells with significant expression of a second polypeptidehaving an amino acid sequence having at least 95% amino acid sequenceidentity to an extracellular domain of the polypeptide having the aminoacid sequence shown in FIG. 4 (SEQ ID NO: 4), lacking its associatedsignal peptide;

(e) significant expression of a polypeptide having an amino acidsequence having at least 95% amino acid sequence identity to apolypeptide encoded by the nucleotide sequence selected from the groupconsisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1) andFIG. 5 (SEQ ID NO: 5), in a minority of the cells or in a population ofcells distinct from cells with significant expression of a secondpolypeptide having an amino acid sequence having at least 95% amino acidsequence identity to a polypeptide encoded by the nucleotide sequenceshown in FIG. 3 (SEQ ID NO: 3); or

(f) significant expression of a polypeptide having an amino acidsequence having at least 95% amino acid sequence identity to apolypeptide encoded by the full-length coding region of the nucleotidesequence selected from the group consisting of the nucleotide sequenceshown in FIG. 1 (SEQ ID NO: 1) and FIG. 5 (SEQ ID NO: 5) in a minorityof the cells or in a population of cells distinct from cells withsignificant expression of a second polypeptide having an amino acidsequence having at least 95% amino acid sequence identity to apolypeptide encoded by the full-length coding region of the nucleotidesequence shown in FIG. 3 (SEQ ID NO: 3),

wherein the identification of said cells from said tumor samplesindicates the identification of a patient for treatment with anantibody, oligopeptide or organic molecule, conjugated to agrowth-inhibitory agent or a cytotoxic agent, said conjugated antibody,oligopeptide or organic molecule binds to said second polypeptide anddepends on internalization into said cells for effective treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) of a TAHO6 (PRO36338)wherein SEQ ID NO:1 is a clone designated herein as “DNA225875” (alsoreferred here in as “CD21” or “CR2”).

FIG. 2 shows the amino acid sequence (SEQ ID NO:2) derived from thecoding sequence of SEQ ID NO:1 shown in FIG. 1.

FIG. 3 shows a nucleotide sequence (SEQ ID NO: 3) of a TAHO25 (PRO36283)cDNA, wherein SEQ ID NO: 3 is a clone designated herein as “DNA225820”(also referred here in as “CD19”).

FIG. 4 shows the amino acid sequence (SEQ ID NO: 4) derived from thecoding sequence of SEQ ID NO: 3 shown in FIG. 3.

FIG. 5 shows a nucleotide sequence (SEQ ID NO: 5) of a TAHO41(PRO283669) cDNA, wherein SEQ ID NO: 5 is a clone designated herein as“DNA548572” (also referred herein as “CD21 variant”)

FIG. 6 shows the amino acid sequence (SEQ ID NO: 6) derived from thecoding sequence of SEQ ID NO: 5 shown in FIG. 5.

FIGS. 7A-7D show microarray data showing the expression of TAHO6 innormal samples and in diseased samples, such as significant expressionin NHL samples and normal lymph node (NLN).

Abbreviations used in the Figures are designated as follows:Non-Hodgkin's Lymphoma (NHL), follicular lymphoma (FL), normal lymphnode (NLN), normal B cells (NB), multiple myeloma cells (MM), smallintestine (s. intestine), fetal liver (f. liver), smooth muscle (s.muscle), fetal brain (f. brain), natural killer cells (NK), neutrophils(N'phil), dendrocytes (DC), memory B cells (mem B), plasma cells (PC),bone marrow plasma cells (BM PC).

FIGS. 8A-8D show microarray data showing the expression of TAHO25 innormal samples and in diseased samples, such as significant expressionin NHL samples, normal lymph node, centroblasts, centrocytes and memoryB cells and in normal tonsil and spleen. Abbreviations used in theFigures are designated as follows: Non-Hodgkin's Lymphoma (NHL),follicular lymphoma (FL), normal lymph node (NLN), normal B cells (NB),multiple myeloma cells (MM), small intestine (s. intestine), fetal liver(f. liver), smooth muscle (s. muscle), fetal brain (f. brain), naturalkiller cells (NK), neutrophils (N'phil), dendrocytes (DC), memory Bcells (mem B), plasma cells (PC), bone marrow plasma cells (BM PC).

FIG. 9 shows surface binding as analyzed by FACS of anti-CD19 (BiomedaCB-19) and anti-CD21 (ATCC HB-135) antibody in various B cell lines,including Daudi, Raji, ARH77, SuDHL4, DoHH2, Ramos, Ramos Clone 1 (Ramoscells expressing CD21 variant (TAHO41)) described in Example 15), RamosClone 2 (Ramos cells expressing CD21 variant (TAHO41)) described inExample 15) and Namalwa cells. Binding is measured as mean fluorescentintensity (MFI).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Definitions

The terms “TAHO polypeptide” and “TAHO” as used herein and whenimmediately followed by a numerical designation, refer to variouspolypeptides, wherein the complete designation (i.e., TAHO/number)refers to specific polypeptide sequences as described herein. The terms“TAHO/number polypeptide” and “TAHO/number” wherein the term “number” isprovided as an actual numerical designation as used herein encompassnative sequence polypeptides, polypeptide variants and fragments ofnative sequence polypeptides and polypeptide variants (which are furtherdefined herein). The TAHO polypeptides described herein may be isolatedfrom a variety of sources, such as from human tissue types or fromanother source, or prepared by recombinant or synthetic methods. Theterm “TAHO polypeptide” refers to each individual TAHO/numberpolypeptide disclosed herein. All disclosures in this specificationwhich refer to the “TAHO polypeptide” refer to each of the polypeptidesindividually as well as jointly. For example, descriptions of thepreparation of, purification of, derivation of, formation of antibodiesto or against, formation of TAHO binding oligopeptides to or against,formation of TAHO binding organic molecules to or against,administration of, compositions containing, treatment of a disease with,etc., pertain to each polypeptide of the invention individually. Theterm “TAHO polypeptide” also includes variants of the TAHO/numberpolypeptides disclosed herein.

“TAHO6” is also herein referred to as “CR2” or “CD21”. “TAHO25” is alsoherein referred to as “CD19”. “TAHO41” is also herein referred to as“CD21 variant” and differs from TAHO6 in a single amino acid at position844. Specifically, amino acid at position 844 of SEQ ID NO: 2 (TAHO6) isa methionine, while amino acid at position 844 of SEQ ID NO: 6 (TAHO41)is an isoleucine.

A “native sequence TAHO polypeptide” comprises a polypeptide having thesame amino acid sequence as the corresponding TAHO polypeptide derivedfrom nature. Such native sequence TAHO polypeptides can be isolated fromnature or can be produced by recombinant or synthetic means. The term“native sequence TAHO polypeptide” specifically encompassesnaturally-occurring truncated or secreted forms of the specific TAHOpolypeptide (e.g., an extracellular domain sequence),naturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of the polypeptide. In certainembodiments of the invention, the native sequence TAHO polypeptidesdisclosed herein are mature or full-length native sequence polypeptidescomprising the full-length amino acids sequences shown in theaccompanying figures. Start and stop codons (if indicated) are shown inbold font and underlined in the figures. Nucleic acid residues indicatedas “N” in the accompanying figures are any nucleic acid residue.However, while the TAHO polypeptides disclosed in the accompanyingfigures are shown to begin with methionine residues designated herein asamino acid position 1 in the figures, it is conceivable and possiblethat other methionine residues located either upstream or downstreamfrom the amino acid position 1 in the figures may be employed as thestarting amino acid residue for the TAHO polypeptides.

The TAHO polypeptide “extracellular domain” or “ECD” refers to a form ofthe TAHO polypeptide which is essentially free of the transmembrane andcytoplasmic domains. Ordinarily, a TAHO polypeptide ECD will have lessthan 1% of such transmembrane and/or cytoplasmic domains and preferably,will have less than 0.5% of such domains. It will be understood that anytransmembrane domains identified for the TAHO polypeptides of thepresent invention are identified pursuant to criteria routinely employedin the art for identifying that type of hydrophobic domain. The exactboundaries of a transmembrane domain may vary but most likely by no morethan about 5 amino acids at either end of the domain as initiallyidentified herein.

Optionally, therefore, an extracellular domain of a TAHO polypeptide maycontain from about 5 or fewer amino acids on either side of thetransmembrane domain/extracellular domain boundary as identified in theExamples or specification and such polypeptides, with or without theassociated signal peptide, and nucleic acid encoding them, arecontemplated by the present invention.

The approximate location of the “signal peptides” of the various TAHOpolypeptides disclosed herein may be shown in the present specificationand/or the accompanying figures. It is noted, however, that theC-terminal boundary of a signal peptide may vary, but most likely by nomore than about 5 amino acids on either side of the signal peptideC-terminal boundary as initially identified herein, wherein theC-terminal boundary of the signal peptide may be identified pursuant tocriteria routinely employed in the art for identifying that type ofamino acid sequence element (e.g., Nielsen et al., Prot. Eng. 10:1-6(1997) and von Heinje et al., Nucl. Acids. Res. 14:4683-4690 (1986)).Moreover, it is also recognized that, in some cases, cleavage of asignal sequence from a secreted polypeptide is not entirely uniform,resulting in more than one secreted species. These mature polypeptides,where the signal peptide is cleaved within no more than about 5 aminoacids on either side of the C-terminal boundary of the signal peptide asidentified herein, and the polynucleotides encoding them, arecontemplated by the present invention.

“TAHO polypeptide variant” means a TAHO polypeptide, preferably anactive TAHO polypeptide, as defined herein having at least about 80%amino acid sequence identity with a full-length native sequence TAHOpolypeptide sequence as disclosed herein, a TAHO polypeptide sequencelacking the signal peptide as disclosed herein, an extracellular domainof a TAHO polypeptide, with or without the signal peptide, as disclosedherein or any other fragment of a full-length TAHO polypeptide sequenceas disclosed herein (such as those encoded by a nucleic acid thatrepresents only a portion of the complete coding sequence for afull-length TAHO polypeptide). Such TAHO polypeptide variants include,for instance, TAHO polypeptides wherein one or more amino acid residuesare added, or deleted, at the N- or C-terminus of the full-length nativeamino acid sequence. Ordinarily, a TAHO polypeptide variant will have atleast about 80% amino acid sequence identity, alternatively at leastabout 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, to afull-length native sequence TAHO polypeptide sequence as disclosedherein, a TAHO polypeptide sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a TAHO polypeptide, with orwithout the signal peptide, as disclosed herein or any otherspecifically defined fragment of a full-length TAHO polypeptide sequenceas disclosed herein. Ordinarily, TAHO variant polypeptides are at leastabout 10 amino acids in length, alternatively at least about 20, 30, 40,50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330,340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470,480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600 aminoacids in length, or more. Optionally, TAHO variant polypeptides willhave no more than one conservative amino acid substitution as comparedto the native TAHO polypeptide sequence, alternatively no more than 2,3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitution ascompared to the native TAHO polypeptide sequence.

“Percent (%) amino acid sequence identity” with respect to the TAHOpolypeptide sequences identified herein is defined as the percentage ofamino acid residues in a candidate sequence that are identical with theamino acid residues in the specific TAHO polypeptide sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the artcan determine appropriate parameters for measuring alignment, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared.

For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2,wherein the complete source code for the ALIGN-2 program is provided inTable 1 below. The ALIGN-2 sequence comparison computer program wasauthored by Genentech, Inc. and the source code shown in Table 1 belowhas been filed with user documentation in the U.S. Copyright Office,Washington D.C., 20559, where it is registered under U.S. CopyrightRegistration No; TXU510087. The ALIGN-2 program is publicly availablethrough Genentech, Inc., South San Francisco, Calif. or may be compiledfrom the source code provided in Table 1 below. The ALIGN-2 programshould be compiled for use on a UNIX operating system, preferablydigital UNIX V4.0D. All sequence comparison parameters are set by theALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. As examples of % amino acid sequence identitycalculations using this method, Tables 2 and 3 demonstrate how tocalculate the % amino acid sequence identity of the amino acid sequencedesignated “Comparison Protein” to the amino acid sequence designated“TAHO”, wherein “TAHO” represents the amino acid sequence of ahypothetical TAHO polypeptide of interest, “Comparison Protein”represents the amino acid sequence of a polypeptide against which the“TAHO” polypeptide of interest is being compared, and “X, “Y” and “Z”each represent different hypothetical amino acid residues. Unlessspecifically stated otherwise, all % amino acid sequence identity valuesused herein are obtained as described in the immediately precedingparagraph using the ALIGN-2 computer program.

“TAHO variant polynucleotide” or “TAHO variant nucleic acid sequence”means a nucleic acid molecule which encodes a TAHO polypeptide,preferably an active TAHO polypeptide, as defined herein and which hasat least about 80% nucleic acid sequence identity with a nucleotide acidsequence encoding a full-length native sequence TAHO polypeptidesequence as disclosed herein, a full-length native sequence TAHOpolypeptide sequence lacking the signal peptide as disclosed herein, anextracellular domain of a TAHO polypeptide, with or without the signalpeptide, as disclosed herein or any other fragment of a full-length TAHOpolypeptide sequence as disclosed herein (such as those encoded by anucleic acid that represents only a portion of the complete codingsequence for a full-length TAHO polypeptide). Ordinarily, a TAHO variantpolynucleotide will have at least about 80% nucleic acid sequenceidentity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%nucleic acid sequence identity with a nucleic acid sequence encoding afull-length native sequence TAHO polypeptide sequence as disclosedherein, a full-length native sequence TAHO polypeptide sequence lackingthe signal peptide as disclosed herein, an extracellular domain of aTAHO polypeptide, with or without the signal sequence, as disclosedherein or any other fragment of a full-length TAHO polypeptide sequenceas disclosed herein. Variants do not encompass the native nucleotidesequence.

Ordinarily, TAHO variant polynucleotides are at least about 5nucleotides in length, alternatively at least about 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110,115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180,185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440,450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580,590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720,730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860,870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000nucleotides in length, wherein in this context the term “about” meansthe referenced nucleotide sequence length plus or minus 10% of thatreferenced length.

“Percent (%) nucleic acid sequence identity” with respect toTAHO-encoding nucleic acid sequences identified herein is defined as thepercentage of nucleotides in a candidate sequence that are identicalwith the nucleotides in the TAHO nucleic acid sequence of interest,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity. Alignment for purposes ofdetermining percent nucleic acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. For purposes herein, however, % nucleicacid sequence identity values are generated using the sequencecomparison computer program ALIGN-2, wherein the complete source codefor the ALIGN-2 program is provided in Table 1 below. The ALIGN-2sequence comparison computer program was authored by Genentech, Inc. andthe source code shown in Table 1 below has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin Table 1 below. The ALIGN-2 program should be compiled for use on aUNIX operating system, preferably digital UNIX V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for nucleic acid sequencecomparisons, the % nucleic acid sequence identity of a given nucleicacid sequence C to, with, or against a given nucleic acid sequence D(which can alternatively be phrased as a given nucleic acid sequence Cthat has or comprises a certain % nucleic acid sequence identity to,with, or against a given nucleic acid sequence D) is calculated asfollows:100 times the fraction W/Zwhere W is the number of nucleotides scored as identical matches by thesequence alignment program ALIGN-2 in that program's alignment of C andD, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C. As examples of % nucleic acid sequence identitycalculations, Tables 4 and 5, demonstrate how to calculate the % nucleicacid sequence identity of the nucleic acid sequence designated“Comparison DNA” to the nucleic acid sequence designated “TAHO-DNA”,wherein “TAHO-DNA” represents a hypothetical TAHO-encoding nucleic acidsequence of interest, “Comparison DNA” represents the nucleotidesequence of a nucleic acid molecule against which the “TAHO-DNA” nucleicacid molecule of interest is being compared, and “N”, “L” and “V” eachrepresent different hypothetical nucleotides. Unless specifically statedotherwise, all % nucleic acid sequence identity values used herein areobtained as described in the immediately preceding paragraph using theALIGN-2 computer program.

In other embodiments, TAHO variant polynucleotides are nucleic acidmolecules that encode a TAHO polypeptide and which are capable ofhybridizing, preferably under stringent hybridization and washconditions, to nucleotide sequences encoding a full-length TAHOpolypeptide as disclosed herein. TAHO variant polypeptides may be thosethat are encoded by a TAHO variant polynucleotide.

The term “full-length coding region” when used in reference to a nucleicacid encoding a TAHO polypeptide refers to the sequence of nucleotideswhich encode the full-length TAHO polypeptide of the invention (which isoften shown between start and stop codons, inclusive thereof, in theaccompanying figures). The term “full-length coding region” when used inreference to an ATCC deposited nucleic acid refers to the TAHOpolypeptide-encoding portion of the cDNA that is inserted into thevector deposited with the ATCC (which is often shown between start andstop codons, inclusive thereof, in the accompanying figures (start andstop codons are bolded and underlined in the figures)).

“Isolated,” when used to describe the various TAHO polypeptidesdisclosed herein, means polypeptide that has been identified andseparated and/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould typically interfere with therapeutic uses for the polypeptide, andmay include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the TAHO polypeptidenatural environment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

An “isolated” TAHO polypeptide-encoding nucleic acid or otherpolypeptide-encoding nucleic acid is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe polypeptide-encoding nucleic acid. An isolated polypeptide-encodingnucleic acid molecule is other than in the form or setting in which itis found in nature. Isolated polypeptide-encoding nucleic acid moleculestherefore are distinguished from the specific polypeptide-encodingnucleic acid molecule as it exists in natural cells. However, anisolated polypeptide-encoding nucleic acid molecule includespolypeptide-encoding nucleic acid molecules contained in cells thatordinarily express the polypeptide where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, may be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)overnight hybridization in a solution that employs 50% formamide, 5×SSC(0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8),0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon spermDNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with a 10minute wash at 42° C. in 0.2×SSC (sodium chloride/sodium citrate)followed by a 10 minute high-stringency wash consisting of 0.1×SSCcontaining EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising a TAHO polypeptide or anti-TAHO antibody fused toa “tag polypeptide”. The tag polypeptide has enough residues to providean epitope against which an antibody can be made, yet is short enoughsuch that it does not interfere with activity of the polypeptide towhich it is fused. The tag polypeptide preferably also is fairly uniqueso that the antibody does not substantially cross-react with otherepitopes. Suitable tag polypeptides generally have at least six aminoacid residues and usually between about 8 and 50 amino acid residues(preferably, between about 10 and 20 amino acid residues).

“Active” or “activity” for the purposes herein refers to form(s) of aTAHO polypeptide which retain a biological and/or an immunologicalactivity of native or naturally-occurring TAHO, wherein “biological”activity refers to a biological function (either inhibitory orstimulatory) caused by a native or naturally-occurring TAHO other thanthe ability to induce the production of an antibody against an antigenicepitope possessed by a native or naturally-occurring TAHO and an“immunological” activity refers to the ability to induce the productionof an antibody against an antigenic epitope possessed by a native ornaturally-occurring TAHO.

The term “antagonist” is used in the broadest sense, and includes anymolecule that partially or fully blocks, inhibits, or neutralizes abiological activity of a native TAHO polypeptide disclosed herein. In asimilar manner, the term “agonist” is used in the broadest sense andincludes any molecule that mimics a biological activity of a native TAHOpolypeptide disclosed herein. Suitable agonist or antagonist moleculesspecifically include agonist or antagonist antibodies or antibodyfragments, fragments or amino acid sequence variants of native TAHOpolypeptides, peptides, antisense oligonucleotides, small organicmolecules, etc. Methods for identifying agonists or antagonists of aTAHO polypeptide may comprise contacting a TAHO polypeptide with acandidate agonist or antagonist molecule and measuring a detectablechange in one or more biological activities normally associated with theTAHO polypeptide.

“Treating” or “treatment” or “alleviation” refers to both therapeutictreatment and prophylactic or preventative measures, wherein the objectis to prevent or slow down (lessen) the targeted pathologic condition ordisorder. Those in need of treatment include those already with thedisorder as well as those prone to have the disorder or those in whomthe disorder is to be prevented. A subject or mammal is successfully“treated” for a TAHO polypeptide-expressing cancer if, after receiving atherapeutic amount of an anti-TAHO antibody, TAHO binding oligopeptideor TAHO binding organic molecule according to the methods of the presentinvention, the patient shows observable and/or measurable reduction inor absence of one or more of the following: reduction in the number ofcancer cells or absence of the cancer cells; reduction in the tumorsize; inhibition (i.e., slow to some extent and preferably stop) ofcancer cell infiltration into peripheral organs including the spread ofcancer into soft tissue and bone; inhibition (i.e., slow to some extentand preferably stop) of tumor metastasis; inhibition, to some extent, oftumor growth; and/or relief to some extent, one or more of the symptomsassociated with the specific cancer; reduced morbidity and mortality,and improvement in quality of life issues. To the extent the anti-TAHOantibody or TAHO binding oligopeptide may prevent growth and/or killexisting cancer cells, it may be cytostatic and/or cytotoxic. Reductionof these signs or symptoms may also be felt by the patient.

The above parameters for assessing successful treatment and improvementin the disease are readily measurable by routine procedures familiar toa physician. For cancer therapy, efficacy can be measured, for example,by assessing the time to disease progression (TTP) and/or determiningthe response rate (RR). Metastasis can be determined by staging testsand by bone scan and tests for calcium level and other enzymes todetermine spread to the bone. CT scans can also be done to look forspread to the pelvis and lymph nodes in the area. Chest X-rays andmeasurement of liver enzyme levels by known methods are used to look formetastasis to the lungs and liver, respectively. Other routine methodsfor monitoring the disease include transrectal ultrasonography (TRUS)and transrectal needle biopsy (TRNB).

For bladder cancer, which is a more localized cancer, methods todetermine progress of disease include urinary cytologic evaluation bycystoscopy, monitoring for presence of blood in the urine, visualizationof the urothelial tract by sonography or an intravenous pyelogram,computed tomography (CT) and magnetic resonance imaging (MRI). Thepresence of distant metastases can be assessed by CT of the abdomen,chest x-rays, or radionuclide imaging of the skeleton.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

“Mammal” for purposes of the treatment of, alleviating the symptoms of acancer refers to any animal classified as a mammal, including humans,domestic and farm animals, and zoo, sports, or pet animals, such asdogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc.Preferably, the mammal is human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

By “solid phase” or “solid support” is meant a non-aqueous matrix towhich an antibody, TAHO binding oligopeptide or TAHO binding organicmolecule of the present invention can adhere or attach. Examples ofsolid phases encompassed herein include those formed partially orentirely of glass (e.g., controlled pore glass), polysaccharides (e.g.,agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones.In certain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g., an affinity chromatography column). This term alsoincludes a discontinuous solid phase of discrete particles, such asthose described in U.S. Pat. No. 4,275,149.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as a TAHO polypeptide, an antibody thereto or a TAHO bindingoligopeptide) to a mammal. The components of the liposome are commonlyarranged in a bilayer formation, similar to the lipid arrangement ofbiological membranes.

A “small” molecule or “small” organic molecule is defined herein to havea molecular weight below about 500 Daltons.

An “effective amount” of a polypeptide, antibody, TAHO bindingoligopeptide, TAHO binding organic molecule or an agonist or antagonistthereof as disclosed herein is an amount sufficient to carry out aspecifically stated purpose. An “effective amount” may be determinedempirically and in a routine manner, in relation to the stated purpose.

The term “therapeutically effective amount” refers to an amount of anantibody, polypeptide, TAHO binding oligopeptide, TAHO binding organicmolecule or other drug effective to “treat” a disease or disorder in asubject or mammal. In the case of cancer, the therapeutically effectiveamount of the drug may reduce the number of cancer cells; reduce thetumor size; inhibit (i.e., slow to some extent and preferably stop)cancer cell infiltration into peripheral organs; inhibit (i.e., slow tosome extent and preferably stop) tumor metastasis; inhibit, to someextent, tumor growth; and/or relieve to some extent one or more of thesymptoms associated with the cancer. See the definition herein of“treating”. To the extent the drug may prevent growth and/or killexisting cancer cells, it may be cytostatic and/or cytotoxic.

A “growth inhibitory amount” of an anti-TAHO antibody, TAHO polypeptide,TAHO binding oligopeptide or TAHO binding organic molecule is an amountcapable of inhibiting the growth of a cell, especially tumor, e.g.,cancer cell, either in vitro or in vivo. A “growth inhibitory amount” ofan anti-TAHO antibody, TAHO polypeptide, TAHO binding oligopeptide orTAHO binding organic molecule for purposes of inhibiting neoplastic cellgrowth may be determined empirically and in a routine manner.

A “cytotoxic amount” of an anti-TAHO antibody, TAHO polypeptide, TAHObinding oligopeptide or TAHO binding organic molecule is an amountcapable of causing the destruction of a cell, especially tumor, e.g.,cancer cell, either in vitro or in vivo. A “cytotoxic amount” of ananti-TAHO antibody, TAHO polypeptide, TAHO binding oligopeptide or TAHObinding organic molecule for purposes of inhibiting neoplastic cellgrowth may be determined empirically and in a routine manner.

The term “antibody” is used in the broadest sense and specificallycovers, for example, single anti-TAHO monoclonal antibodies (includingagonist, antagonist, and neutralizing antibodies), anti-TAHO antibodycompositions with polyepitopic specificity, polyclonal antibodies,single chain anti-TAHO antibodies, and fragments of anti-TAHO antibodies(see below) as long as they exhibit the desired biological orimmunological activity. The term “immunoglobulin” (Ig) is usedinterchangeable with antibody herein.

An “isolated antibody” is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with therapeutic uses for the antibody, and may includeenzymes, hormones, and other proteinaceous or nonproteinaceous solutes.In preferred embodiments, the antibody will be purified (1) to greaterthan 95% by weight of antibody as determined by the Lowry method, andmost preferably more than 99% by weight, (2) to a degree sufficient toobtain at least 15 residues of N-terminal or internal amino acidsequence by use of a spinning cup sequenator, or (3) to homogeneity bySDS-PAGE under reducing or nonreducing conditions using Coomassie blueor, preferably, silver stain. Isolated antibody includes the antibody insitu within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step.

The basic 4-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical light (L) chains and two identical heavy (H)chains (an IgM antibody consists of 5 of the basic heterotetramer unitalong with an additional polypeptide called J chain, and thereforecontain 10 antigen binding sites, while secreted IgA antibodies canpolymerize to form polyvalent assemblages comprising 2-5 of the basic4-chain units along with J chain). In the case of IgGs, the 4-chain unitis generally about 150,000 daltons. Each L chain is linked to a H chainby one covalent disulfide bond, while the two H chains are linked toeach other by one or more disulfide bonds depending on the H chainisotype. Each H and L chain also has regularly spaced intrachaindisulfide bridges. Each H chain has at the N-terminus, a variable domain(V_(H)) followed by three constant domains (C_(H)) for each of the α andγ chains and four C_(H) domains for μ and isotypes. Each L chain has atthe N-terminus, a variable domain (V_(L)) followed by a constant domain(C_(L)) at its other end. The V_(L) is aligned with the V_(H) and theC_(L) is aligned with the first constant domain of the heavy chain(C_(H)1). Particular amino acid residues are believed to form aninterface between the light chain and heavy chain variable domains. Thepairing of a V_(H) and V_(L) together forms a single antigen-bindingsite. For the structure and properties of the different classes ofantibodies, see, e.g., Basic and Clinical Immunology, 8th edition,Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton& Lange, Norwalk, Conn., 1994, page 71 and Chapter 6.

The L chain from any vertebrate species can be assigned to one of twoclearly distinct types, called kappa and lambda, based on the amino acidsequences of their constant domains. Depending on the amino acidsequence of the constant domain of their heavy chains (C_(H)),immunoglobulins can be assigned to different classes or isotypes. Thereare five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, havingheavy chains designated α, δ, ε, γ, and μ, respectively. The γ and αclasses are further divided into subclasses on the basis of relativelyminor differences in C_(H) sequence and function, e.g., humans expressthe following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies. The Vdomain mediates antigen binding and define specificity of a particularantibody for its particular antigen. However, the variability is notevenly distributed across the 110-amino acid span of the variabledomains. Instead, the V regions consist of relatively invariantstretches called framework regions (FRs) of 15-30 amino acids separatedby shorter regions of extreme variability called “hypervariable regions”that are each 9-12 amino acids long. The variable domains of nativeheavy and light chains each comprise four FRs, largely adopting aβ-sheet configuration, connected by three hypervariable regions, whichform loops connecting, and in some cases forming part of, the β-sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (see Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)). The constant domains are not involved directly inbinding an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody dependentcellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. around aboutresidues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the V_(L), and aroundabout 1-35 (H1), 50-65 (H2) and 95-102 (H3) in the V_(H); Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)) and/orthose residues from a “hypervariable loop” (e.g. residues 26-32 (L1),50-52 (L2) and 91-96 (L3) in the V_(L), and 26-32 (H1), 53-55 (H2) and96-101 (H3) in the V_(H); Chothia and Lesk J. Mol. Biol. 196:901-917(1987)).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies useful in the present invention may be prepared by thehybridoma methodology first described by Kohler et al., Nature, 256:495(1975), or may be made using recombinant DNA methods in bacterial,eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567).The “monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991),for example.

The monoclonal antibodies herein include “chimeric” antibodies in whicha portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity (see U.S. Pat. No. 4,816,567; and Morrison et al.,Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies ofinterest herein include “primatized” antibodies comprising variabledomain antigen-binding sequences derived from a non-human primate (e.g.Old World Monkey, Ape etc), and human constant region sequences.

An “intact” antibody is one which comprises an antigen-binding site aswell as a C_(L) and at least heavy chain constant domains, C_(H)1,C_(H)2 and C_(H)3. The constant domains may be native sequence constantdomains (e.g. human native sequence constant domains) or amino acidsequence variant thereof. Preferably, the intact antibody has one ormore effector functions.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870,Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]);single-chain antibody molecules; and multispecific antibodies formedfrom antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, and a residual “Fe” fragment, adesignation reflecting the ability to crystallize readily. The Fabfragment consists of an entire L chain along with the variable regiondomain of the H chain (V_(H)), and the first constant domain of oneheavy chain (C_(H)1). Each Fab fragment is monovalent with respect toantigen binding, i.e., it has a single antigen-binding site. Pepsintreatment of an antibody yields a single large F(ab′)₂ fragment whichroughly corresponds to two disulfide linked Fab fragments havingdivalent antigen-binding activity and is still capable of cross-linkingantigen. Fab′ fragments differ from Fab fragments by having additionalfew residues at the carboxy terminus of the C_(H)1 domain including oneor more cysteines from the antibody hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The Fc fragment comprises the carboxy-terminal portions of both H chainsheld together by disulfides. The effector functions of antibodies aredetermined by sequences in the Fc region, which region is also the partrecognized by Fc receptors (FcR) found on certain types of cells.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervariable loops (3 loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the V_(H) and V_(L) antibody domains connectedinto a single polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the sFv to form the desired structure for antigen binding. For areview of sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994); Borrebaeck 1995, infra.

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10 residues) between the V_(H) and V_(L) domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,resulting in a bivalent fragment, i.e., fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the V_(H) and V_(L) domains of thetwo antibodies are present on different polypeptide chains. Diabodiesare described more fully in, for example, EP 404,097; WO 93/11161; andHollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from the non-humanantibody. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or non-human primate having the desired antibodyspecificity, affinity, and capability. In some instances, frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibodies maycomprise residues that are not found in the recipient antibody or in thedonor antibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

A “species-dependent antibody,” e.g., a mammalian anti-human IgEantibody, is an antibody which has a stronger binding affinity for anantigen from a first mammalian species than it has for a homologue ofthat antigen from a second mammalian species. Normally, thespecies-dependent antibody “bind specifically” to a human antigen (i.e.,has a binding affinity (Kd) value of no more than about 1×10⁻⁷ M,preferably no more than about 1×10⁻⁸ and most preferably no more thanabout 1×10⁻⁹ M) but has a binding affinity for a homologue of theantigen from a second non-human mammalian species which is at leastabout 50 fold, or at least about 500 fold, or at least about 1000 fold,weaker than its binding affinity for the human antigen. Thespecies-dependent antibody can be of any of the various types ofantibodies as defined above, but preferably is a humanized or humanantibody.

A “TAHO binding oligopeptide” is an oligopeptide that binds, preferablyspecifically, to a TAHO polypeptide as described herein. TAHO bindingoligopeptides may be chemically synthesized using known oligopeptidesynthesis methodology or may be prepared and purified using recombinanttechnology. TAHO binding oligopeptides are usually at least about 5amino acids in length, alternatively at least about 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100amino acids in length or more, wherein such oligopeptides that arecapable of binding, preferably specifically, to a TAHO polypeptide asdescribed herein. TAHO binding oligopeptides may be identified withoutundue experimentation using well known techniques. In this regard, it isnoted that techniques for screening oligopeptide libraries foroligopeptides that are capable of specifically binding to a polypeptidetarget are well known in the art (see, e.g., U.S. Pat. Nos. 5,556,762,5,750,373, 4,708,871, 4,833,092, 5,223,409, 5,403,484, 5,571,689,5,663,143; PCT Publication Nos. WO 84/03506 and WO84/03564; Geysen etal., Proc. Natl. Acad. Sci. U.S.A., 81:3998-4002 (1984); Geysen et al.,Proc. Natl. Acad. Sci. U.S.A., 82:178-182 (1985); Geysen et al., inSynthetic Peptides as Antigens, 130-149 (1986); Geysen et al., J.Immunol. Meth., 102:259-274 (1987); Schoofs et al., J. Immunol.,140:611-616 (1988), Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci.USA, 87:6378; Lowman, H. B. et al. (1991) Biochemistry, 30:10832;Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D. et al. (1991),J. Mol. Biol., 222:581; Kang, A. S. et al. (1991) Proc. Natl. Acad. Sci.USA, 88:8363, and Smith, G. P. (1991) Current Opin. Biotechnol., 2:668).

A “TAHO binding organic molecule” is an organic molecule other than anoligopeptide or antibody as defined herein that binds, preferablyspecifically, to a TAHO polypeptide as described herein. TAHO bindingorganic molecules may be identified and chemically synthesized usingknown methodology (see, e.g., PCT Publication Nos. WO00/00823 andWO00/39585). TAHO binding organic molecules are usually less than about2000 daltons in size, alternatively less than about 1500, 750, 500, 250or 200 daltons in size, wherein such organic molecules that are capableof binding, preferably specifically, to a TAHO polypeptide as describedherein may be identified without undue experimentation using well knowntechniques. In this regard, it is noted that techniques for screeningorganic molecule libraries for molecules that are capable of binding toa polypeptide target are well known in the art (see, e.g., PCTPublication Nos. WO00/00823 and WO00/39585).

An antibody, oligopeptide or other organic molecule “which binds” anantigen of interest, e.g. a tumor-associated polypeptide antigen target,is one that binds the antigen with sufficient affinity such that theantibody, oligopeptide or other organic molecule is useful as atherapeutic agent in targeting a cell or tissue expressing the antigen,and does not significantly cross-react with other proteins. In suchembodiments, the extent of binding of the antibody, oligopeptide orother organic molecule to a “non-target” protein will be less than about10% of the binding of the antibody, oligopeptide or other organicmolecule to its particular target protein as determined by fluorescenceactivated cell sorting (FACS) analysis or radioimmunoprecipitation(RIA). With regard to the binding of an antibody, oligopeptide or otherorganic molecule to a target molecule, the term “specific binding” or“specifically binds to” or is “specific for” a particular polypeptide oran epitope on a particular polypeptide target means binding that ismeasurably different from a non-specific interaction. Specific bindingcan be measured, for example, by determining binding of a moleculecompared to binding of a control molecule, which generally is a moleculeof similar structure that does not have binding activity. For example,specific binding can be determined by competition with a controlmolecule that is similar to the target, for example, an excess ofnon-labeled target. In this case, specific binding is indicated if thebinding of the labeled target to a probe is competitively inhibited byexcess unlabeled target. The term “specific binding” or “specificallybinds to” or is “specific for” a particular polypeptide or an epitope ona particular polypeptide target as used herein can be exhibited, forexample, by a molecule having a Kd for the target of at least about 10⁻⁴M, alternatively at least about 10⁻⁵ M, alternatively at least about10⁻⁶ M, alternatively at least about 10⁻⁷ M, alternatively at leastabout 10⁻⁸ M, alternatively at least about 10⁻⁹ M, alternatively atleast about 10⁻¹⁰ M, alternatively at least about 10⁻¹¹ M, alternativelyat least about 10⁻¹² M, or greater. In one embodiment, the term“specific binding” refers to binding where a molecule binds to aparticular polypeptide or epitope on a particular polypeptide withoutsubstantially binding to any other polypeptide or polypeptide epitope.

An antibody, oligopeptide or other organic molecule that “inhibits thegrowth of tumor cells expressing a TAHO polypeptide” or a “growthinhibitory” antibody, oligopeptide or other organic molecule is onewhich results in measurable growth inhibition of cancer cells expressingor overexpressing the appropriate TAHO polypeptide. The TAHO polypeptidemay be a transmembrane polypeptide expressed on the surface of a cancercell or may be a polypeptide that is produced and secreted by a cancercell. Preferred growth inhibitory anti-TAHO antibodies, oligopeptides ororganic molecules inhibit growth of TAHO-expressing tumor cells bygreater than 20%, preferably from about 20% to about 50%, and even morepreferably, by greater than 50% (e.g., from about 50% to about 100%) ascompared to the appropriate control, the control typically being tumorcells not treated with the antibody, oligopeptide or other organicmolecule being tested. In one embodiment, growth inhibition can bemeasured at an antibody concentration of about 0.1 to 30 μg/ml or about0.5 nM to 200 nM in cell culture, where the growth inhibition isdetermined 1-10 days after exposure of the tumor cells to the antibody.Growth inhibition of tumor cells in vivo can be determined in variousways such as is described in the Experimental Examples section below.The antibody is growth inhibitory in vivo if administration of theanti-TAHO antibody at about 1 μg/kg to about 100 mg/kg body weightresults in reduction in tumor size or tumor cell proliferation withinabout 5 days to 3 months from the first administration of the antibody,preferably within about 5 to 30 days.

An antibody, oligopeptide or other organic molecule which “inducesapoptosis” is one which induces programmed cell death as determined bybinding of annexin V, fragmentation of DNA, cell shrinkage, dilation ofendoplasmic reticulum, cell fragmentation, and/or formation of membranevesicles (called apoptotic bodies). The cell is usually one whichoverexpresses a TAHO polypeptide. Preferably the cell is a tumor cell,e.g., a hematopoietic cell, such as a B cell, T cell, basophil,eosinophil, neutrophil, monocyte, platelet or erythrocyte.

Various methods are available for evaluating the cellular eventsassociated with apoptosis. For example, phosphatidyl serine (PS)translocation can be measured by annexin binding; DNA fragmentation canbe evaluated through DNA laddering; and nuclear/chromatin condensationalong with DNA fragmentation can be evaluated by any increase inhypodiploid cells. Preferably, the antibody, oligopeptide or otherorganic molecule which induces apoptosis is one which results in about 2to 50 fold, preferably about 5 to 50 fold, and most preferably about 10to 50 fold, induction of annexin binding relative to untreated cell inan annexin binding assay.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cellactivation.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g., Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The antibodies “arm” the cytotoxiccells and are absolutely required for such killing. The primary cellsfor mediating ADCC, NK cells, express FcγRIII only, whereas monocytesexpress FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cellsis summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.Immunol. 9:457-92 (1991). To assess ADCC activity of a molecule ofinterest, an in vitro ADCC assay, such as that described in U.S. Pat.No. 5,500,362 or 5,821,337 may be performed. Useful effector cells forsuch assays include peripheral blood mononuclear cells (PBMC) andNatural Killer (NK) cells. Alternatively, or additionally, ADCC activityof the molecule of interest may be assessed in vivo, e.g., in a animalmodel such as that disclosed in Clynes et al. (USA) 95:652-656 (1998).

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. The preferred FcR is a native sequence human FcR.Moreover, a preferred FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see review M. inDaëron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed inRavetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991); Capel et al.,Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126:330-41 (1995). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein. The term also includesthe neonatal receptor, FcRn, which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils;with PBMCs and NK cells being preferred. The effector cells may beisolated from a native source, e.g., from blood.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g., as described in Gazzano-Santoro et al.,J. Immunol. Methods 202:163 (1996), may be performed.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include, but are not limitedto, hematopoietic cancers or blood-related cancers, such as lymphoma,leukemia, myeloma or lymphoid malignancies, but also cancers of thespleen and cancers of the lymph nodes. More particular examples of suchB-cell associated cancers, including for example, high, intermediate andlow grade lymphomas (including B cell lymphomas such as, for example,mucosa-associated-lymphoid tissue B cell lymphoma and non-Hodgkin'slymphoma, mantle cell-lymphoma, Burkitt's lymphoma, small lymphocyticlymphoma, marginal zone lymphoma, diffuse large cell lymphoma,follicular lymphoma, and Hodgkin's lymphoma and T cell lymphomas) andleukemias (including secondary leukemia, chronic lymphocytic leukemia,such as B cell leukemia (CD5+B lymphocytes), myeloid leukemia, such asacute myeloid leukemia, chronic myeloid leukemia, lymphoid leukemia,such as acute lymphoblastic leukemia and myelodysplasia), multiplemyeloma, such as plasma cell malignancy, and other hematological and/orB cell- or T-cell-associated cancers. Also included are cancers ofadditional hematopoietic cells, including polymorphonuclear leukocytes,such as basophils, eosinophils, neutrophils and monocytes, dendriticcells, platelets, erythrocytes and natural killer cells. The origins ofB-cell cancers are as follows: marginal zone B-cell lymphoma origins inmemory B-cells in marginal zone, follicular lymphoma and diffuse largeB-cell lymphoma originates in centrocytes in the light zone of germinalcenters, multiple myeloma originates in plasma cells, chroniclymphocytic leukemia and small lymphocytic leukemia originates in B1cells (CD5+), mantle cell lymphoma originates in naive B-cells in themantle zone and Burkitt's lymphoma originates in centroblasts in thedark zone of germinal centers. Tissues which include hematopoietic cellsreferred herein to as “hematopoietic cell tissues” include thymus andbone marrow and peripheral lymphoid tissues, such as spleen, lymphnodes, lymphoid tissues associated with mucosa, such as thegut-associated lymphoid tissues, tonsils, Peyer's patches and appendixand lymphoid tissues associated with other mucosa, for example, thebronchial linings.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one embodiment, the cell proliferative disorder iscancer.

“Tumor”, as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues.

An antibody, oligopeptide or other organic molecule which “induces celldeath” is one which causes a viable cell to become nonviable. The cellis one which expresses a TAHO polypeptide and is of a cell type whichspecifically expresses or overexpresses a TAHO polypeptide. The cell maybe cancerous or normal cells of the particular cell type. The TAHOpolypeptide may be a transmembrane polypeptide expressed on the surfaceof a cancer cell or may be a polypeptide that is produced and secretedby a cancer cell. The cell may be a cancer cell, e.g., a B cell or Tcell. Cell death in vitro may be determined in the absence of complementand immune effector cells to distinguish cell death induced byantibody-dependent cell-mediated cytotoxicity (ADCC) or complementdependent cytotoxicity (CDC). Thus, the assay for cell death may beperformed using heat inactivated serum (i.e., in the absence ofcomplement) and in the absence of immune effector cells. To determinewhether the antibody, oligopeptide or other organic molecule is able toinduce cell death, loss of membrane integrity as evaluated by uptake ofpropidium iodide (PI), trypan blue (see Moore et al. Cytotechnology17:1-11 (1995)) or 7AAD can be assessed relative to untreated cells.Preferred cell death-inducing antibodies, oligopeptides or other organicmolecules are those which induce PI uptake in the PI uptake assay inBT474 cells.

A “TAHO-expressing cell” is a cell which expresses an endogenous ortransfected TAHO polypeptide either on the cell surface or in a secretedform. A “TAHO-expressing cancer” is a cancer comprising cells that havea TAHO polypeptide present on the cell surface or that produce andsecrete a TAHO polypeptide. A “TAHO-expressing cancer” optionallyproduces sufficient levels of TAHO polypeptide on the surface of cellsthereof, such that an anti-TAHO antibody, oligopeptide to other organicmolecule can bind thereto and have a therapeutic effect with respect tothe cancer. In another embodiment, a “TAHO-expressing cancer” optionallyproduces and secretes sufficient levels of TAHO polypeptide, such thatan anti-TAHO antibody, oligopeptide to other organic molecule antagonistcan bind thereto and have a therapeutic effect with respect to thecancer. With regard to the latter, the antagonist may be an antisenseoligonucleotide which reduces, inhibits or prevents production andsecretion of the secreted TAHO polypeptide by tumor cells. A cancerwhich “overexpresses” a TAHO polypeptide is one which has significantlyhigher levels of TAHO polypeptide at the cell surface thereof, orproduces and secretes, compared to a noncancerous cell of the sametissue type. Such overexpression may be caused by gene amplification orby increased transcription or translation. TAHO polypeptideoverexpression may be determined in a detection or prognostic assay byevaluating increased levels of the TAHO protein present on the surfaceof a cell, or secreted by the cell (e.g., via an immunohistochemistryassay using anti-TAHO antibodies prepared against an isolated TAHOpolypeptide which may be prepared using recombinant DNA technology froman isolated nucleic acid encoding the TAHO polypeptide; FACS analysis,etc.). Alternatively, or additionally, one may measure levels of TAHOpolypeptide-encoding nucleic acid or mRNA in the cell, e.g., viafluorescent in situ hybridization using a nucleic acid based probecorresponding to a TAHO-encoding nucleic acid or the complement thereof;(FISH; see WO98/45479 published October, 1998), Southern blotting,Northern blotting, or polymerase chain reaction (PCR) techniques, suchas real time quantitative PCR (RT-PCR). One may also study TAHOpolypeptide overexpression by measuring shed antigen in a biologicalfluid such as serum, e.g, using antibody-based assays (see also, e.g.,U.S. Pat. No. 4,933,294 issued Jun. 12, 1990; WO91/05264 published Apr.18, 1991; U.S. Pat. No. 5,401,638 issued Mar. 28, 1995; and Sias et al.,J. Immunol. Methods 132:73-80 (1990)). Aside from the above assays,various in vivo assays are available to the skilled practitioner. Forexample, one may expose cells within the body of the patient to anantibody which is optionally labeled with a detectable label, e.g., aradioactive isotope, and binding of the antibody to cells in the patientcan be evaluated, e.g., by external scanning for radioactivity or byanalyzing a biopsy taken from a patient previously exposed to theantibody.

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibody,oligopeptide or other organic molecule so as to generate a “labeled”antibody, oligopeptide or other organic molecule. The label may bedetectable by itself (e.g. radioisotope labels or fluorescent labels)or, in the case of an enzymatic label, may catalyze chemical alterationof a substrate compound or composition which is detectable.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g.,At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents e.g. methotrexate, adriamicin,vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,melphalan, mitomycin C, chlorambucil, daunorubicin or otherintercalating agents, enzymes and fragments thereof such as nucleolyticenzymes, antibiotics, and toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof, and the variousantitumor or anticancer agents disclosed below. Other cytotoxic agentsare described below. A tumoricidal agent causes destruction of tumorcells.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell, especially aTAHO-expressing cancer cell, either in vitro or in vivo. Thus, thegrowth inhibitory agent may be one which significantly reduces thepercentage of TAHO-expressing cells in S phase. Examples of growthinhibitory agents include agents that block cell cycle progression (at aplace other than S phase), such as agents that induce G1 arrest andM-phase arrest. Classical M-phase blockers include the vincas(vincristine and vinblastine), taxanes, and topoisomerase II inhibitorssuch as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.Those agents that arrest G1 also spill over into S-phase arrest, forexample, DNA alkylating agents such as tamoxifen, prednisone,dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil,and ara-C. Further information can be found in The Molecular Basis ofCancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycleregulation, oncogenes, and antineoplastic drugs” by Murakami et al. (W BSaunders: Philadelphia, 1995), especially p. 13. The taxanes (paclitaxeland docetaxel) are anticancer drugs both derived from the yew tree.Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the Europeanyew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-MyersSquibb). Paclitaxel and docetaxel promote the assembly of microtubulesfrom tubulin dimers and stabilize microtubules by preventingdepolymerization, which results in the inhibition of mitosis in cells.

“Doxorubicin” is an anthracycline antibiotic. The full chemical name ofdoxorubicin is(8S-cis)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexapyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione.

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such as TNF-α orTNF-β; and other polypeptide factors including LIF and kit ligand (KL).As used herein, the term cytokine includes proteins from natural sourcesor from recombinant cell culture and biologically active equivalents ofthe native sequence cytokines.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts. TABLE 2 TAHO XXXXXXXXXXXXXXX (Length = 15 amino acids)Comparison XXXXXYYYYYYY (Length = 12 amino acids) Protein% amino acid sequence identity = (the number of identically matchingamino acid residues between the two polypeptide sequences as determinedby ALIGN-2) divided by (the total number of amino acid residues of theTAHO polypeptide) = 5 divided by 15 = 33.3%

TABLE 3 TAHO XXXXXXXXXX (Length = 10 amino acids) ComparisonXXXXXYYYYYYZZYZ (Length = 15 amino acids) Protein% amino acid sequence identity = (the number of identically matchingamino acid residues between the two polypeptide sequences as determinedby ALIGN-2) divided by (the total number of amino acid residues of theTAHO polypeptide) = 5 divided by 10 = 50%

TABLE 4 TAHO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides) ComparisonNNNNNNLLLLLLLLLL (Length = 16 nucleotides) DNA% nucleic acid sequence identity = (the number of identically matchingnucleotides between the two nucleic acid sequences as determined byALIGN-2) divided by (the total number of nucleotides of the TAHO-DNAnucleic acid sequence) = 6 divided by 14 = 42.9%

TABLE 5 TAHO-DNA NNNNNNNNNNNN (Length = 12 nucleotides) Comparison DNANNNNLLLVV (Length = 9 nucleotides)% nucleic acid sequence identity = (the number of identically matchingnucleotides between the two nucleic acid sequences as determined byALIGN-2) divided by (the total number of nucleotides of the TAHO-DNAnucleic acid sequence) = 4 divided by 12 = 33.3%II. Compositions and Methods of the Invention

A. Anti-TAHO Antibodies

In one embodiment, the present invention provides anti-TAHO antibodieswhich may find use herein as therapeutic agents. Exemplary antibodiesinclude polyclonal, monoclonal, humanized, bispecific, andheteroconjugate antibodies.

1. Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen (especially when synthetic peptides are used) to a protein thatis immunogenic in the species to be immunized. For example, the antigencan be conjugated to keyhole limpet hemocyanin (KLH), serum albumin,bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctionalor derivatizing agent, e.g., maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysine residues), glutaraldehyde, succinic anhydride, SOCl₂, orR¹N═C═NR, where R and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later, the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later, theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. Conjugates also can be made inrecombinant cell culture as protein fusions. Also, aggregating agentssuch as alum are suitably used to enhance the immune response.

2. Monoclonal Antibodies

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as described above to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the protein used for immunization. Alternatively, lymphocytesmay be immunized in vitro. After immunization, lymphocytes are isolatedand then fused with a myeloma cell line using a suitable fusing agent,such as polyethylene glycol, to form a hybridoma cell (Goding,Monoclonal Antibodies: Principles and Practice, pp. 59-103 (AcademicPress, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium which medium preferably contains one or more substancesthat inhibit the growth or survival of the unfused, parental myelomacells (also referred to as fusion partner). For example, if the parentalmyeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the selective culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred fusion partner myeloma cells are those that fuse efficiently,support stable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a selective medium thatselects against the unfused parental cells. Preferred myeloma cell linesare murine myeloma lines, such as those derived from MOPC-21 and MPC-11mouse tumors available from the Salk Institute Cell Distribution Center,San Diego, Calif. USA, and SP-2 and derivatives e.g., X63-Ag8-653 cellsavailable from the American Type Culture Collection, Manassas, Va., USA.Human myeloma and mouse-human heteromyeloma cell lines also have beendescribed for the production of human monoclonal antibodies (Kozbor, J.Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunosorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis described in Munson et al., Anal.Biochem., 107:220 (1980).

Once hybridoma cells that produce antibodies of the desired specificity,affinity, and/or activity are identified, the clones may be subcloned bylimiting dilution procedures and grown by standard methods (Goding,Monoclonal Antibodies: Principles and Practice, pp. 59-103 (AcademicPress, 1986)). Suitable culture media for this purpose include, forexample, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells maybe grown in vivo as ascites tumors in an animal e.g, by i.p. injectionof the cells into mice.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional antibody purification procedures such as, for example,affinity chromatography (e.g., using protein A or protein G-Sepharose)or ion-exchange chromatography, hydroxylapatite chromatography, gelelectrophoresis, dialysis, etc.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, ormyeloma cells that do not otherwise produce antibody protein, to obtainthe synthesis of monoclonal antibodies in the recombinant host cells.Review articles on recombinant expression in bacteria of DNA encodingthe antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262(1993) and Pluckthun, Immunol. Revs. 130:151-188 (1992).

In a further embodiment, monoclonal antibodies or antibody fragments canbe isolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348:552-554 (1990). Clackson etal., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol.,222:581-597 (1991) describe the isolation of murine and humanantibodies, respectively, using phage libraries. Subsequent publicationsdescribe the production of high affinity (nM range) human antibodies bychain shuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), aswell as combinatorial infection and in vivo recombination as a strategyfor constructing very large phage libraries (Waterhouse et al., Nuc.Acids. Res. 21:2265-2266 (1993%). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

The DNA that encodes the antibody may be modified to produce chimeric orfusion antibody polypeptides, for example, by substituting human heavychain and light chain constant domain (C_(H) and C_(L)) sequences forthe homologous murine sequences (U.S. Pat. No. 4,816,567; and Morrison,et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by fusing theimmunoglobulin coding sequence with all or part of the coding sequencefor a non-immunoglobulin polypeptide (heterologous polypeptide). Thenon-immunoglobulin polypeptide sequences can substitute for the constantdomains of an antibody, or they are substituted for the variable domainsof one antigen-combining site of an antibody to create a chimericbivalent antibody comprising one antigen-combining site havingspecificity for an antigen and another antigen-combining site havingspecificity for a different antigen.

3. Human and Humanized Antibodies

The anti-TAHO antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ Or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity and HAMA response (human anti-mouse antibody) when theantibody is intended for human therapeutic use. According to theso-called “best-fit” method, the sequence of the variable domain of arodent antibody is screened against the entire library of known humanvariable domain sequences. The human V domain sequence which is closestto that of the rodent is identified and the human framework region (FR)within it accepted for the humanized antibody (Sims et al., J. Immunol.151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). Anothermethod uses a particular framework region derived from the consensussequence of all human antibodies of a particular subgroup of light orheavy chains. The same framework may be used for several differenthumanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285(1992); Presta et al., J. Immunol. 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh binding affinity for the antigen and other favorable biologicalproperties. To achieve this goal, according to a preferred method,humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art.

Computer programs are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind its antigen. In thisway, FR residues can be selected and combined from the recipient andimport sequences so that the desired antibody characteristic, such asincreased affinity for the target antigen(s), is achieved. In general,the hypervariable region residues are directly and most substantiallyinvolved in influencing antigen binding.

Various forms of a humanized anti-TAHO antibody are contemplated. Forexample, the humanized antibody may be an antibody fragment, such as aFab, which is optionally conjugated with one or more cytotoxic agent(s)in order to generate an immunoconjugate. Alternatively, the humanizedantibody may be an intact antibody, such as an intact IgG1 antibody.

As an alternative to humanization, human antibodies can be generated.For example, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array into such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann etal., Year in Immuno. 7:33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825,5,591,669 (all of GenPharm); 5,545,807; and WO 97/17852.

Alternatively, phage display technology (McCafferty et al., Nature348:552-553 [1990]) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats, reviewed in, e.g., Johnson, Kevin S, and Chiswell,David J., Current Opinion in Structural Biology 3:564-571 (1993).Several sources of V-gene segments can be used for phage display.Clackson et al., Nature, 352:624-628 (1991) isolated a diverse array ofanti-oxazolone antibodies from a small random combinatorial library of Vgenes derived from the spleens of immunized mice. A repertoire of Vgenes from unimmunized human donors can be constructed and antibodies toa diverse array of antigens (including self-antigens) can be isolatedessentially following the techniques described by Marks et al., J. Mol.Biol. 222:581-597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993).See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905.

As discussed above, human antibodies may also be generated by in vitroactivated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

4. Antibody Fragments

In certain circumstances there are advantages of using antibodyfragments, rather than whole antibodies. The smaller size of thefragments allows for rapid clearance, and may lead to improved access tosolid tumors.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al., Bio/Technology 10:163-167 (1992)). According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab′)₂ fragment with increased in vivohalf-life comprising a salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. In other embodiments, the antibody of choice is a singlechain Fv fragment (scFv). See WO 93/16185; U.S. Pat. No. 5,571,894; andU.S. Pat. No. 5,587,458. Fv and sFv are the only species with intactcombining sites that are devoid of constant regions; thus, they aresuitable for reduced nonspecific binding during in vivo use. sFv fusionproteins may be constructed to yield fusion of an effector protein ateither the amino or the carboxy terminus of an sFv. See AntibodyEngineering, ed. Borrebaeck, supra. The antibody fragment may also be a“linear antibody”, e.g., as described in U.S. Pat. No. 5,641,870 forexample. Such linear antibody fragments may be monospecific orbispecific.

5. Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities forat least two different epitopes. Exemplary bispecific antibodies maybind to two different epitopes of a TAHO protein as described herein.Other such antibodies may combine a TAHO binding site with a bindingsite for another protein. Alternatively, an anti-TAHO arm may becombined with an arm which binds to a triggering molecule on a leukocytesuch as a T-cell receptor molecule (e.g. CD3), or Fc receptors for IgG(FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16), so as tofocus and localize cellular defense mechanisms to the TAHO-expressingcell. Bispecific antibodies may also be used to localize cytotoxicagents to cells which express TAHO. These antibodies possess aTAHO-binding arm and an arm which binds the cytotoxic agent (e.g.,saporin, anti-interferon-α, vinca alkaloid, ricin A chain, methotrexateor radioactive isotope hapten). Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g., F(ab′)₂ bispecificantibodies).

WO 96/16673 describes a bispecific anti-ErbB2/anti-FcγRIII antibody andU.S. Pat. No. 5,837,234 discloses a bispecific anti-ErbB2/anti-FcγRIantibody. A bispecific anti-ErbB2/Fcα antibody is shown in WO98/02463.U.S. Pat. No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3antibody.

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe co-expression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ. 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. Preferably, thefusion is with an Ig heavy chain constant domain, comprising at leastpart of the hinge, C_(H)2, and C_(H)3 regions. It is preferred to havethe first heavy-chain constant region (C_(H)1) containing the sitenecessary for light chain bonding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable host cell.This provides for greater flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yield of the desired bispecific antibody. It is,however, possible to insert the coding sequences for two or all threepolypeptide chains into a single expression vector when the expressionof at least two polypeptide chains in equal ratios results in highyields or when the ratios have no significant affect on the yield of thedesired chain combination.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology 121:210 (1986).

According to another approach described in U.S. Pat. No. 5,731,168, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the C_(H)3 domain. In this method, one or more small amino acidside chains from the interface of the first antibody molecule arereplaced with larger side chains (e.g., tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g., alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science 229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent, sodiumarsenite, to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med. 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the ErbB2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets. Various techniques for making and isolatingbispecific antibody fragments directly from recombinant cell culturehave also been described. For example, bispecific antibodies have beenproduced using leucine zippers. Kostelny et al., J. Immunol.148(5):1547-1553 (1992). The leucine zipper peptides from the Fos andJun proteins were linked to the Fab′ portions of two differentantibodies by gene fusion. The antibody homodimers were reduced at thehinge region to form monomers and then re-oxidized to form the antibodyheterodimers. This method can also be utilized for the production ofantibody homodimers. The “diabody” technology described by Hollinger etal., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided analternative mechanism for making bispecific antibody fragments. Thefragments comprise a V_(H) connected to a V_(L) by a linker which is tooshort to allow pairing between the two domains on the same chain.

Accordingly, the V_(H) and V_(L) domains of one fragment are forced topair with the complementary V_(L) and V_(H) domains of another fragment,thereby forming two antigen-binding sites. Another strategy for makingbispecific antibody fragments by the use of single-chain Fv (sFv) dimershas also been reported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60(1991).

6. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells [U.S. Pat. No. 4,676,980],and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP03089]. It is contemplated that the antibodies may be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

7. Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The antibodies of the present invention can bemultivalent antibodies (which are other than of the IgM class) withthree or more antigen binding sites (e.g. tetravalent antibodies), whichcan be readily produced by recombinant expression of nucleic acidencoding the polypeptide chains of the antibody. The multivalentantibody can comprise a dimerization domain and three or more antigenbinding sites. The preferred dimerization domain comprises (or consistsof) an Fc region or a hinge region. In this scenario, the antibody willcomprise an Fc region and three or more antigen binding sitesamino-terminal to the Fc region. The preferred multivalent antibodyherein comprises (or consists of) three to about eight, but preferablyfour, antigen binding sites. The multivalent antibody comprises at leastone polypeptide chain (and preferably two polypeptide chains), whereinthe polypeptide chain(s) comprise two or more variable domains. Forinstance, the polypeptide chain(s) may compriseVD1-(X1)_(n)-VD2-(X2)_(n)-Fc,

wherein VD1 is a first variable domain, VD2 is a second variable domain,Fc is one polypeptide chain of an Fc region, X1 and X2 represent anamino acid or polypeptide, and n is 0 or 1. For instance, thepolypeptide chain(s) may comprise: VH-CH1-flexible linker-VH-CH1-Fcregion chain; or VH-CH1-VH-CH1-Fc region chain. The multivalent antibodyherein preferably further comprises at least two (and preferably four)light chain variable domain polypeptides. The multivalent antibodyherein may, for instance, comprise from about two to about eight lightchain variable domain polypeptides. The light chain variable domainpolypeptides contemplated here comprise a light chain variable domainand, optionally, further comprise a CL domain.

8. Effector Function Engineering

It may be desirable to modify the antibody of the invention with respectto effector function, e.g., so as to enhance antigen-dependentcell-mediated cyotoxicity (ADCC) and/or complement dependentcytotoxicity (CDC) of the antibody. This may be achieved by introducingone or more amino acid substitutions in an Fc region of the antibody.Alternatively or additionally, cysteine residue(s) may be introduced inthe Fc region, thereby allowing interchain disulfide bond formation inthis region. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). See Caronet al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol.148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumoractivity may also be prepared using heterobifunctional cross-linkers asdescribed in Wolff et al., Cancer Research 53:2560-2565 (1993).Alternatively, an antibody can be engineered which has dual Fc regionsand may thereby have enhanced complement lysis and ADCC capabilities.See Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989). Toincrease the serum half life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment) as described in U.S. Pat. No. 5,739,277, for example.As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, orIgG₄) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

9. Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a chemotherapeutic agent, agrowth inhibitory agent, a toxin (e.g., an enzymatically active toxin ofbacterial, fungal, plant, or animal origin, or fragments thereof), or aradioactive isotope (i.e., a radioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re. Conjugates of the antibody and cytotoxic agent are made usinga variety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such asbis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

Conjugates of an antibody and one or more small molecule toxins, such asa calicheamicin, auristatin peptides, such as monomethylauristatin(MMAE) (synthetic analog of dolastatin), maytansinoids, such as DM1, atrichothene, and CC1065, and the derivatives of these toxins that havetoxin activity, are also contemplated herein.

Maytansine and Maytansinoids

In one preferred embodiment, an anti-TAHO antibody (full length orfragments) of the invention is conjugated to one or more maytansinoidmolecules.

Maytansinoids, such as DM1, are mitototic inhibitors which act byinhibiting tubulin polymerization. Maytansine was first isolated fromthe east African shrub Maytenus serrata (U.S. Pat. No. 3,896,111).Subsequently, it was discovered that certain microbes also producemaytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat.No. 4,151,042). Synthetic maytansinol and derivatives and analoguesthereof are disclosed, for example, in U.S. Pat. Nos. 4,137,230;4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016;4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821;4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254;4,362,663; and 4,371,533, the disclosures of which are hereby expresslyincorporated by reference.

Maytansinoid-Antibody Conjugates

In an attempt to improve their therapeutic index, maytansine andmaytansinoids have been conjugated to antibodies specifically binding totumor cell antigens. Immunoconjugates containing maytansinoids and theirtherapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235 B1, the disclosures of whichare hereby expressly incorporated by reference. Liu et al., Proc. Natl.Acad. Sci. USA 93:8618-8623 (1996) described immunoconjugates comprisinga maytansinoid designated DM1 linked to the monoclonal antibody C242directed against human colorectal cancer. The conjugate was found to behighly cytotoxic towards cultured colon cancer cells, and showedantitumor activity in an in vivo tumor growth assay. Chari et al.,Cancer Research 52:127-131 (1992) describe immunoconjugates in which amaytansinoid was conjugated via a disulfide linker to the murineantibody A7 binding to an antigen on human colon cancer cell lines, orto another murine monoclonal antibody TA.1 that binds the HER-2/neuoncogene. The cytotoxicity of the TA.1-maytansonoid conjugate was testedin vitro on the human breast cancer cell line SK-BR-3, which expresses3×10⁵HER-2 surface antigens per cell. The drug conjugate achieved adegree of cytotoxicity similar to the free maytansonid drug, which couldbe increased by increasing the number of maytansinoid molecules perantibody molecule. The A7-maytansinoid conjugate showed low systemiccytotoxicity in mice.

Anti-TAHO Polypeptide Antibody-Maytansinoid Conjugates(Immunoconjugates)

Anti-TAHO antibody-maytansinoid conjugates are prepared by chemicallylinking an anti-TAHO antibody to a maytansinoid molecule withoutsignificantly diminishing the biological activity of either the antibodyor the maytansinoid molecule. An average of 3-4 maytansinoid moleculesconjugated per antibody molecule has shown efficacy in enhancingcytotoxicity of target cells without negatively affecting the functionor solubility of the antibody, although even one molecule oftoxin/antibody would be expected to enhance cytotoxicity over the use ofnaked antibody. Maytansinoids are well known in the art and can besynthesized by known techniques or isolated from natural sources.Suitable maytansinoids are disclosed, for example, in U.S. Pat. No.5,208,020 and in the other patents and nonpatent publications referredto hereinabove. Preferred maytansinoids are maytansinol and maytansinolanalogues modified in the aromatic ring or at other positions of themaytansinol molecule, such as various maytansinol esters.

There are many linking groups known in the art for makingantibody-maytansinoid conjugates, including, for example, thosedisclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, andChari et al., Cancer Research 52:127-131 (1992). The linking groupsinclude disulfide groups, thioether groups, acid labile groups,photolabile groups, peptidase labile groups, or esterase labile groups,as disclosed in the above-identified patents, disulfide and thioethergroups being preferred.

Conjugates of the antibody and maytansinoid may be made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas his (p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agentsinclude N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson etal., Biochem. J. 173:723-737 [1978]), sulfosuccinimidyl maleimidomethylcyclohexane carboxylate (SMCC) andN-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for adisulfide linkage. Other useful linkers include cys-MC-vc-PAB (avaline-citrulline (vc) dipeptide linker reagent having a maleimidecomponent and a para-aminobenzylcarbamoyl (PAB) self-immolativecomponent.

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhyrdoxymethyl, the C-15 position modified with a hydroxyl group, and theC-20 position having a hydroxyl group. In a preferred embodiment, thelinkage is formed at the C-3 position of maytansinol or a maytansinolanalogue.

Calicheamicin

Another immunoconjugate of interest comprises an anti-TAHO antibodyconjugated to one or more calicheamicin molecules. The calicheamicinfamily of antibiotics are capable of producing double-stranded DNAbreaks at sub-picomolar concentrations. For the preparation ofconjugates of the calicheamicin family, see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001,5,877,296 (all to American Cyanamid Company). Structural analogues ofcalicheamicin which may be used include, but are not limited to, γ₁^(I), α₂ ^(I), α₃ ^(I), N-acetyl-γ₁ ^(I), PSAG and θ^(I) ₁ (Hinman etal., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research58:2925-2928 (1998) and the aforementioned U.S. patents to AmericanCyanamid). Another anti-tumor drug that the antibody can be conjugatedis QFA which is an antifolate. Both calicheamicin and QFA haveintracellular sites of action and do not readily cross the plasmamembrane. Therefore, cellular uptake of these agents through antibodymediated internalization greatly enhances their cytotoxic effects.

Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the anti-TAHOantibodies of the invention include BCNU, streptozoicin, vincristine and5-fluorouracil, the family of agents known collectively LL-E33288complex described in U.S. Pat. Nos. 5,053,394, 5,770,710, as well asesperamicins (U.S. Pat. No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an immunoconjugate formedbetween an antibody and a compound with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated anti-TAHO antibodies. Examplesinclude At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹²and radioactive isotopes of Lu. When the conjugate is used fordetection, it may comprise a radioactive atom for scintigraphic studies,for example tc^(99m) or I¹²³, or a spin label for nuclear magneticresonance (NMR) imaging (also known as magnetic resonance imaging, mri),such as iodine-123 again, iodine-131, indium-111, fluorine-19,carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as tc^(99m) or I¹²³, Re¹⁸⁶ Re¹⁸⁸ and In¹¹¹ can be attachedvia a cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas his (p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

Alternatively, a fusion protein comprising the anti-TAHO antibody andcytotoxic agent may be made, e.g., by recombinant techniques or peptidesynthesis. The length of DNA may comprise respective regions encodingthe two portions of the conjugate either adjacent one another orseparated by a region encoding a linker peptide which does not destroythe desired properties of the conjugate.

In yet another embodiment, the antibody may be conjugated to a“receptor” (such streptavidin) for utilization in tumor pre-targetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g., avidin)which is conjugated to a cytotoxic agent (e.g., a radionucleotide).

10. Immunoliposomes

The anti-TAHO antibodies disclosed herein may also be formulated asimmunoliposomes. A “liposome” is a small vesicle composed of varioustypes of lipids, phospholipids and/or surfactant which is useful fordelivery of a drug to a mammal. The components of the liposome arecommonly arranged in a bilayer formation, similar to the lipidarrangement of biological membranes. Liposomes containing the antibodyare prepared by methods known in the art, such as described in Epsteinet al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc.Natl. Acad. Sci. USA 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and4,544,545; and WO97/38731 published Oct. 23, 1997. Liposomes withenhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.

Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem. 257:286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent is optionally contained within the liposome. SeeGabizon et al., J. National Cancer Inst. 81(19):1484 (1989).

B. TAHO Binding Oligopeptides

TAHO binding oligopeptides of the present invention are oligopeptidesthat bind, preferably specifically, to a TAHO polypeptide as describedherein. TAHO binding oligopeptides may be chemically synthesized usingknown oligopeptide synthesis methodology or may be prepared and purifiedusing recombinant technology. TAHO binding oligopeptides are usually atleast about 5 amino acids in length, alternatively at least about 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or 100 amino acids in length or more, wherein such oligopeptidesthat are capable of binding, preferably specifically, to a TAHOpolypeptide as described herein. TAHO binding oligopeptides may beidentified without undue experimentation using well known techniques. Inthis regard, it is noted that techniques for screening oligopeptidelibraries for oligopeptides that are capable of specifically binding toa polypeptide target are well known in the art (see, e.g., U.S. Pat.Nos. 5,556,762, 5,750,373, 4,708,871, 4,833,092, 5,223,409, 5,403,484,5,571,689, 5,663,143; PCT Publication Nos. WO 84/03506 and WO84/03564;Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 81:3998-4002 (1984);Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 82:178-182 (1985); Geysenet al., in Synthetic Peptides as Antigens, 130-149 (1986); Geysen etal., J. Immunol. Meth., 102:259-274 (1987); Schoofs et al., J. Immunol.,140:611-616 (1988), Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci.USA, 87:6378; Lowman, H. B. et al. (1991) Biochemistry, 30:10832;Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D. et al. (1991),J. Mol. Biol., 222:581; Kang, A. S. et al. (1991) Proc. Natl. Acad. Sci.USA, 88:8363, and Smith, G. P. (1991) Current Opin. Biotechnol., 2:668).

In this regard, bacteriophage (phage) display is one well knowntechnique which allows one to screen large oligopeptide libraries toidentify member(s) of those libraries which are capable of specificallybinding to a polypeptide target. Phage display is a technique by whichvariant polypeptides are displayed as fusion proteins to the coatprotein on the surface of bacteriophage particles (Scott, J. K. andSmith, G. P. (1990) Science, 249: 386). The utility of phage displaylies in the fact that large libraries of selectively randomized proteinvariants (or randomly cloned cDNAs) can be rapidly and efficientlysorted for those sequences that bind to a target molecule with highaffinity. Display of peptide (Cwirla, S. E. et al. (1990) Proc. Natl.Acad. Sci. USA, 87:6378) or protein (Lowman, H. B. et al. (1991)Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352: 624;Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. et al.(1991) Proc. Natl. Acad. Sci. USA, 88:8363) libraries on phage have beenused for screening millions of polypeptides or oligopeptides for oneswith specific binding properties (Smith, G. P. (1991) Current Opin.Biotechnol., 2:668). Sorting phage libraries of random mutants requiresa strategy for constructing and propagating a large number of variants,a procedure for affinity purification using the target receptor, and ameans of evaluating the results of binding enrichments. U.S. Pat. Nos.5,223,409, 5,403,484, 5,571,689, and 5,663,143.

Although most phage display methods have used filamentous phage,lambdoid phage display systems (WO 95/34683; U.S. Pat. No. 5,627,024),T4 phage display systems (Ren et al., Gene, 215: 439 (1998); Zhu et al.,Cancer Research, 58(15): 3209-3214 (1998); Jiang et al., Infection &Immunity, 65(11): 4770-4777 (1997); Ren et al., Gene, 195(2):303-311(1997); Ren, Protein Sci., 5: 1833 (1996); Efimov et al., Virus Genes,10: 173 (1995)) and T7 phage display systems (Smith and Scott, Methodsin Enzymology, 217: 228-257 (1993); U.S. Pat. No. 5,766,905) are alsoknown.

Many other improvements and variations of the basic phage displayconcept have now been developed. These improvements enhance the abilityof display systems to screen peptide libraries for binding to selectedtarget molecules and to display functional proteins with the potentialof screening these proteins for desired properties. Combinatorialreaction devices for phage display reactions have been developed (WO98/14277) and phage display libraries have been used to analyze andcontrol bimolecular interactions (WO 98/20169; WO 98/20159) andproperties of constrained helical peptides (WO 98/20036). WO 97/35196describes a method of isolating an affinity ligand in which a phagedisplay library is contacted with one solution in which the ligand willbind to a target molecule and a second solution in which the affinityligand will not bind to the target molecule, to selectively isolatebinding ligands. WO 97/46251 describes a method of biopanning a randomphage display library with an affinity purified antibody and thenisolating binding phage, followed by a micropanning process usingmicroplate wells to isolate high affinity binding phage. The use ofStaphylococcus aureus protein A as an affinity tag has also beenreported (Li et al. (1998) Mol. Biotech., 9:187). WO 97/47314 describesthe use of substrate subtraction libraries to distinguish enzymespecificities using a combinatorial library which may be a phage displaylibrary. A method for selecting enzymes suitable for use in detergentsusing phage display is described in WO 97/09446. Additional methods ofselecting specific binding proteins are described in U.S. Pat. Nos.5,498,538, 5,432,018, and WO 98/15833.

Methods of generating peptide libraries and screening these librariesare also disclosed in U.S. Pat. Nos. 5,723,286, 5,432,018, 5,580,717,5,427,908, 5,498,530, 5,770,434, 5,734,018, 5,698,426, 5,763,192, and5,723,323.

C. TAHO Binding Organic Molecules

TAHO binding organic molecules are organic molecules other thanoligopeptides or antibodies as defined herein that bind, preferablyspecifically, to a TAHO polypeptide as described herein. TAHO bindingorganic molecules may be identified and chemically synthesized usingknown methodology (see, e.g., PCT Publication Nos. WO00/00823 andWO00/39585). TAHO binding organic molecules are usually less than about2000 daltons in size, alternatively less than about 1500, 750, 500, 250or 200 daltons in size, wherein such organic molecules that are capableof binding, preferably specifically, to a TAHO polypeptide as describedherein may be identified without undue experimentation using well knowntechniques. In this regard, it is noted that techniques for screeningorganic molecule libraries for molecules that are capable of binding toa polypeptide target are well known in the art (see, e.g., PCTPublication Nos. WO00/00823 and WO00/39585). TAHO binding organicmolecules may be, for example, aldehydes, ketones, oximes, hydrazones,semicarbazones, carbazides, primary amines, secondary amines, tertiaryamines, N-substituted hydrazines, hydrazides, alcohols, ethers, thiols,thioethers, disulfides, carboxylic acids, esters, amides, ureas,carbamates, carbonates, ketals, thioketals, acetals, thioacetals, arylhalides, aryl sulfonates, alkyl halides, alkyl sulfonates, aromaticcompounds, heterocyclic compounds, anilines, alkenes, alkynes, diols,amino alcohols, oxazolidines, oxazolines, thiazolidines, thiazolines,enamines, sulfonamides, epoxides, aziridines, isocyanates, sulfonylchlorides, diazo compounds, acid chlorides, or the like.

D. Screening for Anti-TAHO Antibodies, TAHO Binding Oligopeptides andTAHO Binding Organic Molecules with the Desired Properties

Techniques for generating antibodies, oligopeptides and organicmolecules that bind to TAHO polypeptides have been described above. Onemay further select antibodies, oligopeptides or other organic moleculeswith certain biological characteristics, as desired.

The growth inhibitory effects of an anti-TAHO antibody, oligopeptide orother organic molecule of the invention may be assessed by methods knownin the art, e.g., using cells which express a TAHO polypeptide eitherendogenously or following transfection with the TAHO gene. For example,appropriate tumor cell lines and TAHO-transfected cells may be treatedwith an anti-TAHO monoclonal antibody, oligopeptide or other organicmolecule of the invention at various concentrations for a few days(e.g., 2-7) days and stained with crystal violet or MTT or analyzed bysome other colorimetric assay. Another method of measuring proliferationwould be by comparing H-thymidine uptake by the cells treated in thepresence or absence an anti-TAHO antibody, TAHO binding oligopeptide orTAHO binding organic molecule of the invention. After treatment, thecells are harvested and the amount of radioactivity incorporated intothe DNA quantitated in a scintillation counter. Appropriate positivecontrols include treatment of a selected cell line with a growthinhibitory antibody known to inhibit growth of that cell line. Growthinhibition of tumor cells in vivo can be determined in various waysknown in the art. The tumor cell may be one that overexpresses a TAHOpolypeptide. The anti-TAHO antibody, TAHO binding oligopeptide or TAHObinding organic molecule will inhibit cell proliferation of aTAHO-expressing tumor cell in vitro or in vivo by about 25-100% comparedto the untreated tumor cell, more preferably, by about 30-100%, and evenmore preferably by about 50-100% or 70-100%, in one embodiment, at anantibody concentration of about 0.5 to 30 μg/ml. Growth inhibition canbe measured at an antibody concentration of about 0.5 to 30 μg/ml orabout 0.5 nM to 200 nM in cell culture, where the growth inhibition isdetermined 1-10 days after exposure of the tumor cells to the antibody.The antibody is growth inhibitory in vivo if administration of theanti-TAHO antibody at about 1 μg/kg to about 100 mg/kg body weightresults in reduction in tumor size or reduction of tumor cellproliferation within about 5 days to 3 months from the firstadministration of the antibody, preferably within about 5 to 30 days.

To select for an anti-TAHO antibody, TAHO binding oligopeptide or TAHObinding organic molecule which induces cell death, loss of membraneintegrity as indicated by, e.g., propidium iodide (PI), trypan blue or7AAD uptake may be assessed relative to control. A PI uptake assay canbe performed in the absence of complement and immune effector cells.TAHO polypeptide-expressing tumor cells are incubated with medium aloneor medium containing the appropriate anti-TAHO antibody (e.g, at about10 μg/1 ml), TAHO binding oligopeptide or TAHO binding organic molecule.The cells are incubated for a 3 day time period.

Following each treatment, cells are washed and aliquoted into 35 mmstrainer-capped 12×75 tubes (1 ml per tube, 3 tubes per treatment group)for removal of cell clumps. Tubes then receive PI (10 μg/ml). Samplesmay be analyzed using a FACSCAN® flow cytometer and FACSCONVERT®CellQuest software (Becton Dickinson). Those anti-TAHO antibodies, TAHObinding oligopeptides or TAHO binding organic molecules that inducestatistically significant levels of cell death as determined by PIuptake may be selected as cell death-inducing anti-TAHO antibodies, TAHObinding oligopeptides or TAHO binding organic molecules.

To screen for antibodies, oligopeptides or other organic molecules whichbind to an epitope on a TAHO polypeptide bound by an antibody ofinterest, a routine cross-blocking assay such as that described inAntibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, EdHarlow and David Lane (1988), can be performed. This assay can be usedto determine if a test antibody, oligopeptide or other organic moleculebinds the same site or epitope as a known anti-TAHO antibody.Alternatively, or additionally, epitope mapping can be performed bymethods known in the art. For example, the antibody sequence can bemutagenized such as by alanine scanning, to identify contact residues.The mutant antibody is initially tested for binding with polyclonalantibody to ensure proper folding. In a different method, peptidescorresponding to different regions of a TAHO polypeptide can be used incompetition assays with the test antibodies or with a test antibody andan antibody with a characterized or known epitope.

E. Antibody Dependent Enzyme Mediated Prodrug Therapy (ADEPT)

The antibodies of the present invention may also be used in ADEPT byconjugating the antibody to a prodrug-activating enzyme which converts aprodrug (e.g., a peptidyl chemotherapeutic agent, see WO81/01145) to anactive anti-cancer drug. See, for example, WO 88/07378 and U.S. Pat. No.4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includesany enzyme capable of acting on a prodrug in such a way so as to covertit into its more active, cytotoxic form.

Enzymes that are useful in the method of this invention include, but arenot limited to, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug, 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidaseuseful for converting glycosylated prodrugs into free drugs; β-lactamaseuseful for converting drugs derivatized with β-lactams into free drugs;and penicillin amidases, such as penicillin V amidase or penicillin Gamidase, useful for converting drugs derivatized at their aminenitrogens with phenoxyacetyl or phenylacetyl groups, respectively, intofree drugs. Alternatively, antibodies with enzymatic activity, alsoknown in the art as “abzymes”, can be used to convert the prodrugs ofthe invention into free active drugs (see, e.g., Massey, Nature328:457-458 (1987)). Antibody-abzyme conjugates can be prepared asdescribed herein for delivery of the abzyme to a tumor cell population.

The enzymes of this invention can be covalently bound to the anti-TAHOantibodies by techniques well known in the art such as the use of theheterobifunctionaL crosslinking reagents discussed above. Alternatively,fusion proteins comprising at least the antigen binding region of anantibody of the invention linked to at least a functionally activeportion of an enzyme of the invention can be constructed usingrecombinant DNA techniques well known in the art (see, e.g., Neubergeret al., Nature 312:604-608 (1984).

F. Full-Length TAHO Polypeptides

The present invention also provides newly identified and isolatednucleotide sequences encoding polypeptides referred to in the presentapplication as TAHO polypeptides. In particular, cDNAs (partial andfull-length) encoding various TAHO polypeptides have been identified andisolated, as disclosed in further detail in the Examples below.

As disclosed in the Examples below, various cDNA clones have beendeposited with the ATCC. The actual nucleotide sequences of those clonescan readily be determined by the skilled artisan by sequencing of thedeposited clone using routine methods in the art. The predicted aminoacid sequence can be determined from the nucleotide sequence usingroutine skill. For the TAHO polypeptides and encoding nucleic acidsdescribed herein, in some cases, Applicants have identified what isbelieved to be the reading frame best identifiable with the sequenceinformation available at the time.

G. Anti-TAHO Antibody and TAHO Polypeptide Variants

In addition to the anti-TAHO antibodies and full-length native sequenceTAHO polypeptides described herein, it is contemplated that anti-TAHOantibody and TAHO polypeptide variants can be prepared. Anti-TAHOantibody and TAHO polypeptide variants can be prepared by introducingappropriate nucleotide changes into the encoding DNA, and/or bysynthesis of the desired antibody or polypeptide. Those skilled in theart will appreciate that amino acid changes may alter post-translationalprocesses of the anti-TAHO antibody or TAHO polypeptide, such aschanging the number or position of glycosylation sites or altering themembrane anchoring characteristics.

Variations in the anti-TAHO antibodies and TAHO polypeptides describedherein, can be made, for example, using any of the techniques andguidelines for conservative and non-conservative mutations set forth,for instance, in U.S. Pat. No. 5,364,934. Variations may be asubstitution, deletion or insertion of one or more codons encoding theantibody or polypeptide that results in a change in the amino acidsequence as compared with the native sequence antibody or polypeptide.Optionally the variation is by substitution of at least one amino acidwith any other amino acid in one or more of the domains of the anti-TAHOantibody or TAHO polypeptide. Guidance in determining which amino acidresidue may be inserted, substituted or deleted without adverselyaffecting the desired activity may be found by comparing the sequence ofthe anti-TAHO antibody or TAHO polypeptide with that of homologous knownprotein molecules and minimizing the number of amino acid sequencechanges made in regions of high homology. Amino acid substitutions canbe the result of replacing one amino acid with another amino acid havingsimilar structural and/or chemical properties, such as the replacementof a leucine with a serine, i.e., conservative amino acid replacements.Insertions or deletions may optionally be in the range of about 1 to 5amino acids. The variation allowed may be determined by systematicallymaking insertions, deletions or substitutions of amino acids in thesequence and testing the resulting variants for activity exhibited bythe full-length or mature native sequence.

Anti-TAHO antibody and TAHO polypeptide fragments are provided herein.Such fragments may be truncated at the N-terminus or C-terminus, or maylack internal residues, for example, when compared with a full lengthnative antibody or protein. Certain fragments lack amino acid residuesthat are not essential for a desired biological activity of theanti-TAHO antibody or TAHO polypeptide.

Anti-TAHO antibody and TAHO polypeptide fragments may be prepared by anyof a number of conventional techniques. Desired peptide fragments may bechemically synthesized. An alternative approach involves generatingantibody or polypeptide fragments by enzymatic digestion, e.g., bytreating the protein with an enzyme known to cleave proteins at sitesdefined by particular amino acid residues, or by digesting the DNA withsuitable restriction enzymes and isolating the desired fragment. Yetanother suitable technique involves isolating and amplifying a DNAfragment encoding a desired antibody or polypeptide fragment, bypolymerase chain reaction (PCR). Oligonucleotides that define thedesired termini of the DNA fragment are employed at the 5′ and 3′primers in the PCR. Preferably, anti-TAHO antibody and TAHO polypeptidefragments share at least one biological and/or immunological activitywith the native anti-TAHO antibody or TAHO polypeptide disclosed herein.

In particular embodiments, conservative substitutions of interest areshown in Table 6 under the heading of preferred substitutions. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated exemplary substitutions in Table 6, oras further described below in reference to amino acid classes, areintroduced and the products screened. TABLE 6 Original Residue ExemplarySubstitutions Preferred Substitutions Ala (A) val; leu; ile val Arg (R)lys; gln; asn lys Asn (N) gln; his; lys; arg gln Asp (D) glu glu Cys (C)ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His (H)asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe; leu norleucineLeu (L) norleucine; ile; val; ile met; ala; phe Lys (K) arg; gln; asnarg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr leu Pro(P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y)trp; phe; thr; ser phe Val (V) ile; leu; met; phe; leu ala; norleucine

Substantial modifications in function or immunological identity of theanti-TAHO antibody or TAHO polypeptide are accomplished by selectingsubstitutions that differ significantly in their effect on maintaining(a) the structure of the polypeptide backbone in the area of thesubstitution, for example, as a sheet or helical conformation, (b) thecharge or hydrophobicity of the molecule at the target site, or (c) thebulk of the side chain. Naturally occurring residues are divided intogroups based on common side-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gin, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the anti-TAHO antibody or TAHO polypeptidevariant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant [Cunningham and Wells,Science, 244:1081-1085 (1989)]. Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions [Creighton, The Proteins,(W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

Any cysteine residue not involved in maintaining the proper conformationof the anti-TAHO antibody or TAHO polypeptide also may be substituted,generally with serine, to improve the oxidative stability of themolecule and prevent aberrant crosslinking. Conversely, cysteine bond(s)may be added to the anti-TAHO antibody or TAHO polypeptide to improveits stability (particularly where the antibody is an antibody fragmentsuch as an Fv fragment).

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g., a humanized or human antibody). Generally, the resultingvariant(s) selected for further development will have improvedbiological properties relative to the parent antibody from which theyare generated. A convenient way for generating such substitutionalvariants involves affinity maturation using phage display. Briefly,several hypervariable region sites (e.g., 6-7 sites) are mutated togenerate all possible amino substitutions at each site. The antibodyvariants thus generated are displayed in a monovalent fashion fromfilamentous phage particles as fusions to the gene III product of M13packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g., binding affinity) asherein disclosed. In order to identify candidate hypervariable regionsites for modification, alanine scanning mutagenesis can be performed toidentify hypervariable region residues contributing significantly toantigen binding. Alternatively, or additionally, it may be beneficial toanalyze a crystal structure of the antigen-antibody complex to identifycontact points between the antibody and human TAHO polypeptide. Suchcontact residues and neighboring residues are candidates forsubstitution according to the techniques elaborated herein. Once suchvariants are generated, the panel of variants is subjected to screeningas described herein and antibodies with superior properties in one ormore relevant assays may be selected for further development.

Nucleic acid molecules encoding amino acid sequence variants of theanti-TAHO antibody are prepared by a variety of methods known in theart. These methods include, but are not limited to, isolation from anatural source (in the case of naturally occurring amino acid sequencevariants) or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared variant or a non-variant version of the anti-TAHO antibody.

H. Modifications of Anti-TAHO Antibodies and TAHO Polypeptides

Covalent modifications of anti-TAHO antibodies and TAHO polypeptides areincluded within the scope of this invention. One type of covalentmodification includes reacting targeted amino acid residues of ananti-TAHO antibody or TAHO polypeptide with an organic derivatizingagent that is capable of reacting with selected side chains or the N- orC-terminal residues of the anti-TAHO antibody or TAHO polypeptide.Derivatization with bifunctional agents is useful, for instance, forcrosslinking anti-TAHO antibody or TAHO polypeptide to a water-insolublesupport matrix or surface for use in the method for purifying anti-TAHOantibodies, and vice-versa. Commonly used crosslinking agents include,e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the anti-TAHO antibody or TAHOpolypeptide included within the scope of this invention comprisesaltering the native glycosylation pattern of the antibody orpolypeptide. “Altering the native glycosylation pattern” is intended forpurposes herein to mean deleting one or more carbohydrate moieties foundin native sequence anti-TAHO antibody or TAHO polypeptide (either byremoving the underlying glycosylation site or by deleting theglycosylation by chemical and/or enzymatic means), and/or adding one ormore glycosylation sites that are not present in the native sequenceanti-TAHO antibody or TAHO polypeptide. In addition, the phrase includesqualitative changes in the glycosylation of the native proteins,involving a change in the nature and proportions of the variouscarbohydrate moieties present.

Glycosylation of antibodies and other polypeptides is typically eitherN-linked or O-linked. N-linked refers to the attachment of thecarbohydrate moiety to the side chain of an asparagine residue. Thetripeptide sequences asparagine-X-serine and asparagine-X-threonine,where X is any amino acid except proline, are the recognition sequencesfor enzymatic attachment of the carbohydrate moiety to the asparagineside chain. Thus, the presence of either of these tripeptide sequencesin a polypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the anti-TAHO antibody or TAHOpolypeptide is conveniently accomplished by altering the amino acidsequence such that it contains one or more of the above-describedtripeptide sequences (for N-linked glycosylation sites). The alterationmay also be made by the addition of, or substitution by, one or moreserine or threonine residues to the sequence of the original anti-TAHOantibody or TAHO polypeptide (for O-linked glycosylation sites). Theanti-TAHO antibody or TAHO polypeptide amino acid sequence mayoptionally be altered through changes at the DNA level, particularly bymutating the DNA encoding the anti-TAHO antibody or TAHO polypeptide atpreselected bases such that codons are generated that will translateinto the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theanti-TAHO antibody or TAHO polypeptide is by chemical or enzymaticcoupling of glycosides to the polypeptide. Such methods are described inthe art, e.g., in WO 87/05330 published 11 Sep. 1987, and in Aplin andWriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the anti-TAHO antibody orTAHO polypeptide may be accomplished chemically or enzymatically or bymutational substitution of codons encoding for amino acid residues thatserve as targets for glycosylation. Chemical deglycosylation techniquesare known in the art and described, for instance, by Hakimuddin, et al.,Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal.Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol.,138:350 (1987).

Another type of covalent modification of anti-TAHO antibody or TAHOpolypeptide comprises linking the antibody or polypeptide to one of avariety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG),polypropylene glycol, or polyoxyalkylenes, in the manner set forth inU.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or4,179,337. The antibody or polypeptide also may be entrapped inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization (for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules), or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed.,(1980).

The anti-TAHO antibody or TAHO polypeptide of the present invention mayalso be modified in a way to form chimeric molecules comprising ananti-TAHO antibody or TAHO polypeptide fused to another, heterologouspolypeptide or amino acid sequence.

In one embodiment, such a chimeric molecule comprises a fusion of theanti-TAHO antibody or TAHO polypeptide with a tag polypeptide whichprovides an epitope to which an anti-tag antibody can selectively bind.The epitope tag is generally placed at the amino- or carboxyl-terminusof the anti-TAHO antibody or TAHO polypeptide. The presence of suchepitope-tagged forms of the anti-TAHO antibody or TAHO polypeptide canbe detected using an antibody against the tag polypeptide. Also,provision of the epitope tag enables the anti-TAHO antibody or TAHOpolypeptide to be readily purified by affinity purification using ananti-tag antibody or another type of affinity matrix that binds to theepitope tag. Various tag polypeptides and their respective antibodiesare well known in the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192-194 (1992)]; an α-tubulin epitope peptide [Skinneret al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)].

In an alternative embodiment, the chimeric molecule may comprise afusion of the anti-TAHO antibody or TAHO polypeptide with animmunoglobulin or a particular region of an immunoglobulin. For abivalent form of the chimeric molecule (also referred to as an“immunoadhesin”), such a fusion could be to the Fc region of an IgGmolecule. The Ig fusions preferably include the substitution of asoluble (transmembrane domain deleted or inactivated) form of ananti-TAHO antibody or TAHO polypeptide in place of at least one variableregion within an Ig molecule. In a particularly preferred embodiment,the immunoglobulin fusion includes the hinge, CH₂ and CH₃, or the hinge,CH₁, CH₂ and CH₃ regions of an IgG1 molecule. For the production ofimmunoglobulin fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27,1995.

I. Preparation of Anti-TAHO Antibodies and TAHO Polypeptides

The description below relates primarily to production of anti-TAHOantibodies and TAHO polypeptides by culturing cells transformed ortransfected with a vector containing anti-TAHO antibody- and TAHOpolypeptide-encoding nucleic acid. It is, of course, contemplated thatalternative methods, which are well known in the art, may be employed toprepare anti-TAHO antibodies and TAHO polypeptides. For instance, theappropriate amino acid sequence, or portions thereof, may be produced bydirect peptide synthesis using solid-phase techniques [see, e.g.,Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., SanFrancisco, Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154(1963)]. In vitro protein synthesis may be performed using manualtechniques or by automation. Automated synthesis may be accomplished,for instance, using an Applied Biosystems Peptide Synthesizer (FosterCity, Calif.) using manufacturer's instructions. Various portions of theanti-TAHO antibody or TAHO polypeptide may be chemically synthesizedseparately and combined using chemical or enzymatic methods to producethe desired anti-TAHO antibody or TAHO polypeptide.

1. Isolation of DNA Encoding Anti-TAHO Antibody or TAHO Polypeptide

DNA encoding anti-TAHO antibody or TAHO polypeptide may be obtained froma cDNA library prepared from tissue believed to possess the anti-TAHOantibody or TAHO polypeptide mRNA and to express it at a detectablelevel. Accordingly, human anti-TAHO antibody or TAHO polypeptide DNA canbe conveniently obtained from a cDNA library prepared from human tissue.The anti-TAHO antibody- or TAHO polypeptide-encoding gene may also beobtained from a genomic library or by known synthetic procedures (e.g.,automated nucleic acid synthesis).

Libraries can be screened with probes (such as oligonucleotides of atleast about 20-80 bases) designed to identify the gene of interest orthe protein encoded by it. Screening the cDNA or genomic library withthe selected probe may be conducted using standard procedures, such asdescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual(New York: Cold Spring Harbor Laboratory Press, 1989). An alternativemeans to isolate the gene encoding anti-TAHO antibody or TAHOpolypeptide is to use PCR methodology [Sambrook et al., supra;Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring HarborLaboratory Press, 1995)].

Techniques for screening a cDNA library are well known in the art. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., supra.

Sequences identified in such library screening methods can be comparedand aligned to other known sequences deposited and available in publicdatabases such as GenBank or other private sequence databases. Sequenceidentity (at either the amino acid or nucleotide level) within definedregions of the molecule or across the full-length sequence can bedetermined using methods known in the art and as described herein.

Nucleic acid having protein coding sequence may be obtained by screeningselected cDNA or genomic libraries using the deduced amino acid sequencedisclosed herein for the first time, and, if necessary, usingconventional primer extension procedures as described in Sambrook etal., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

2. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for anti-TAHO antibody or TAHO polypeptideproduction and cultured in conventional nutrient media modified asappropriate for inducing promoters, selecting transformants, oramplifying the genes encoding the desired sequences. The cultureconditions, such as media, temperature, pH and the like, can be selectedby the skilled artisan without undue experimentation. In general,principles, protocols, and practical techniques for maximizing theproductivity of cell cultures can be found in Mammalian CellBiotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991)and Sambrook et al., supra.

Methods of eukaryotic cell transfection and prokaryotic celltransformation are known to the ordinarily skilled artisan, for example,CaCl₂, CaPO₄, liposome-mediated and electroporation. Depending on thehost cell used, transformation is performed using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride, as described in Sambrook et al., supra, or electroporation isgenerally used for prokaryotes. Infection with Agrobacterium tumefaciensis used for transformation of certain plant cells, as described by Shawet al., Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransfections have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K₁₂ strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting. Strain W3110 is one particularly preferred host or parenthost because it is a common host strain for recombinant DNA productfermentations. Preferably, the host cell secretes minimal amounts ofproteolytic enzymes. For example, strain W3110 may be modified to effecta genetic mutation in the genes encoding proteins endogenous to thehost, with examples of such hosts including E. coli W3110 strain 1A2,which has the complete genotype tonA; E. coli W3110 strain 9E4, whichhas the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC55,244), which has the complete genotype tonA ptr3 phoA E15(argF-lac)169 degP ompT kan^(r) ; E. coli W3110 strain 37D6, which hasthe complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7ilvG kan^(r) ; E. coli W3110 strain 40B4, which is strain 37D6 with anon-kanamycin resistant degP deletion mutation; and an E. coli strainhaving mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783issued 7 Aug. 1990. Alternatively, in vitro methods of cloning, e.g.,PCR or other nucleic acid polymerase reactions, are suitable.

Full length antibody, antibody fragments, and antibody fusion proteinscan be produced in bacteria, in particular when glycosylation and Fceffector function are not needed, such as when the therapeutic antibodyis conjugated to a cytotoxic agent (e.g., a toxin) and theimmunoconjugate by itself shows effectiveness in tumor cell destruction.Full length antibodies have greater half life in circulation. Productionin E. coli is faster and more cost efficient. For expression of antibodyfragments and polypeptides in bacteria, see, e.g., U.S. Pat. No.5,648,237 (Carter et. al.), U.S. Pat. No. 5,789,199 (Joly et al.), andU.S. Pat. No. 5,840,523 (Simmons et al.) which describes translationinitiation regio (TIR) and signal sequences for optimizing expressionand secretion, these patents incorporated herein by reference. Afterexpression, the antibody is isolated from the E. coli cell paste in asoluble fraction and can be purified through, e.g., a protein A or Gcolumn depending on the isotype. Final purification can be carried outsimilar to the process for purifying antibody expressed e.g, in CHOcells.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for anti-TAHOantibody- or TAHO polypeptide-encoding vectors. Saccharomyces cerevisiaeis a commonly used lower eukaryotic host microorganism. Others includeSchizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 [1981]; EP139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Pat. No.4,943,529; Fleer et al., Bio/Technology, 9:968-975 (1991)) such as,e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J.Bacteriol., 154(2):737-742 [1983]), K. fragilis (ATCC 12,424), K.bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al.,Bio/Technology, 8:135 (1990)), K. thermotolerans, and K. marxianus;yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al.,J. Basic Microbiol., 28:265-278 [1988]); Candida; Trichoderma reesia (EP244,234); Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA,76:5259-5263 [1979]); Schwanniomyces such as Schwanniomyces occidentalis(EP 394,538 published 31 Oct. 1990); and filamentous fungi such as,e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10Jan. 1991), and Aspergillus hosts such as A. nidulans (Ballance et al.,Biochem. Biophys. Res. Commun., 112:284-289 [1983]; Tilburn et al.,Gene, 26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475-479[1985]). Methylotropic yeasts are suitable herein and include, but arenot limited to, yeast capable of growth on methanol selected from thegenera consisting of Hansenula, Candida, Kloeckera, Pichia,Saccharomyces, Torulopsis, and Rhodotorula. A list of specific speciesthat are exemplary of this class of yeasts may be found in C. Anthony,The Biochemistry of Methylotrophs, 269 (1982).

Suitable host cells for the expression of glycosylated anti-TAHOantibody or TAHO polypeptide are derived from multicellular organisms.Examples of invertebrate cells include insect cells such as DrosophilaS2 and Spodoptera Sf9, as well as plant cells, such as cell cultures ofcotton, corn, potato, soybean, petunia, tomato, and tobacco. Numerousbaculoviral strains and variants and corresponding permissive insecthost cells from hosts such as Spodoptera frugiperda (caterpillar), Aedesaegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to thepresent invention, particularly for transfection of Spodopterafrugiperda cells.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/−DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for anti-TAHO antibody or TAHO polypeptide productionand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

3. Selection and Use of a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding anti-TAHO antibodyor TAHO polypeptide may be inserted into a replicable vector for cloning(amplification of the DNA) or for expression. Various vectors arepublicly available. The vector may, for example, be in the form of aplasmid, cosmid, viral particle, or phage. The appropriate nucleic acidsequence may be inserted into the vector by a variety of procedures. Ingeneral, DNA is inserted into an appropriate restriction endonucleasesite(s) using techniques known in the art. Vector components generallyinclude, but are not limited to, one or more of a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence. Construction ofsuitable vectors containing one or more of these components employsstandard ligation techniques which are known to the skilled artisan.

The TAHO may be produced recombinantly not only directly, but also as afusion polypeptide with a heterologous polypeptide, which may be asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. In general, thesignal sequence may be a component of the vector, or it may be a part ofthe anti-TAHO antibody- or TAHO polypeptide-encoding DNA that isinserted into the vector. The signal sequence may be a prokaryoticsignal sequence selected, for example, from the group of the alkalinephosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders.For yeast secretion the signal sequence may be, e.g., the yeastinvertase leader, alpha factor leader (including Saccharomyces andKluyveromyces α-factor leaders, the latter described in U.S. Pat. No.5,010,182), or acid phosphatase leader, the C. albicans glucoamylaseleader (EP 362,179 published 4 Apr. 1990), or the signal described in WO90/13646 published 15 Nov. 1990. In mammalian cell expression, mammaliansignal sequences may be used to direct secretion of the protein, such assignal sequences from secreted polypeptides of the same or relatedspecies, as well as viral secretory leaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2μ plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up theanti-TAHO antibody- or TAHO polypeptide-encoding nucleic acid, such asDHFR or thymidine kinase. An appropriate host cell when wild-type DHFRis employed is the CHO cell line deficient in DHFR activity, preparedand propagated as described by Urlaub et al., Proc. Natl. Acad. Sci.USA, 77:4216 (1980). A suitable selection gene for use in yeast is thetrp1 gene present in the yeast plasmid YRp7 [Stinchcomb et al., Nature,282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al.,Gene, 10:157 (1980)]. The trp1 gene provides a selection marker for amutant strain of yeast lacking the ability to grow in tryptophan, forexample, ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)].

Expression and cloning vectors usually contain a promoter operablylinked to the anti-TAHO antibody- or TAHO polypeptide-encoding nucleicacid sequence to direct mRNA synthesis. Promoters recognized by avariety of potential host cells are well known. Promoters suitable foruse with prokaryotic hosts include the β-lactamase and lactose promotersystems [Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature,281:544 (1979)], alkaline phosphatase, a tryptophan (trp) promotersystem [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], andhybrid promoters such as the tac promoter [deBoer et al., Proc. Natl.Acad. Sci. USA, 80:21-25 (1983)]. Promoters for use in bacterial systemsalso will contain a Shine-Dalgarno (S.D.) sequence operably linked tothe DNA encoding anti-TAHO antibody or TAHO polypeptide.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

Anti-TAHO antibody or TAHO polypeptide transcription from vectors inmammalian host cells is controlled, for example, by promoters obtainedfrom the genomes of viruses such as polyoma virus, fowlpox virus (UK2,211,504 published 5 Jul. 1989), adenovirus (such as Adenovirus 2),bovine papilloma virus, avian sarcoma virus, cytomegalovirus, aretrovirus, hepatitis-B virus and Simian Virus 40 (SV40), fromheterologous mammalian promoters, e.g., the actin promoter or animmunoglobulin promoter, and from heat-shock promoters, provided suchpromoters are compatible with the host cell systems.

Transcription of a DNA encoding the anti-TAHO antibody or TAHOpolypeptide by higher eukaryotes may be increased by inserting anenhancer sequence into the vector. Enhancers are cis-acting elements ofDNA, usually about from 10 to 300 bp, that act on a promoter to increaseits transcription. Many enhancer sequences are now known from mammaliangenes (globin, elastase, albumin, α-fetoprotein, and insulin).Typically, however, one will use an enhancer from a eukaryotic cellvirus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. The enhancer may be spliced into thevector at a position 5′ or 3′ to the anti-TAHO antibody or TAHOpolypeptide coding sequence, but is preferably located at a site 5′ fromthe promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding anti-TAHO antibody or TAHO polypeptide.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of anti-TAHO antibody or TAHO polypeptide in recombinantvertebrate cell culture are described in Gething et al., Nature,293:620-625 (1981); Mantei et al., Nature, 281:40-46 (1979); EP 117,060;and EP 117,058.

4. Culturing the Host Cells

The host cells used to produce the anti-TAHO antibody or TAHOpolypeptide of this invention may be cultured in a variety of media.Commercially available media such as Ham's F10 (Sigma), MinimalEssential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco'sModified Eagle's Medium ((DMEM), Sigma) are suitable for culturing thehost cells. In addition, any of the media described in Ham et al., Meth.Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S.Pat. No. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be used as culturemedia for the host cells. Any of these media may be supplemented asnecessary with hormones and/or other growth factors (such as insulin,transferrin, or epidermal growth factor), salts (such as sodiumchloride, calcium, magnesium, and phosphate), buffers (such as HEPES),nucleotides (such as adenosine and thymidine), antibiotics (such asGENTAMYCIN™ drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

5. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequenceTAHO polypeptide or against a synthetic peptide based on the DNAsequences provided herein or against exogenous sequence fused to TAHODNA and encoding a specific antibody epitope.

6. Purification of Anti-TAHO Antibody and TAHO Polypeptide

Forms of anti-TAHO antibody and TAHO polypeptide may be recovered fromculture medium or from host cell lysates. If membrane-bound, it can bereleased from the membrane using a suitable detergent solution (e.g.Triton-X 100) or by enzymatic cleavage. Cells employed in expression ofanti-TAHO antibody and TAHO polypeptide can be disrupted by variousphysical or chemical means, such as freeze-thaw cycling, sonication,mechanical disruption, or cell lysing agents.

It may be desired to purify anti-TAHO antibody and TAHO polypeptide fromrecombinant cell proteins or polypeptides. The following procedures areexemplary of suitable purification procedures: by fractionation on anion-exchange column; ethanol precipitation; reverse phase HPLC;chromatography on silica or on a cation-exchange resin such as DEAE;chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gelfiltration using, for example, Sephadex G-75; protein A Sepharosecolumns to remove contaminants such as IgG; and metal chelating columnsto bind epitope-tagged forms of the anti-TAHO antibody and TAHOpolypeptide. Various methods of protein purification may be employed andsuch methods are known in the art and described for example inDeutscher, Methods in Enzymology, 182 (1990); Scopes, ProteinPurification: Principles and Practice, Springer-Verlag, New York (1982).The purification step(s) selected will depend, for example, on thenature of the production process used and the particular anti-TAHOantibody or TAHO polypeptide produced.

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, areremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10:163-167 (1992) describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2 or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a C_(H)3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

J. Pharmaceutical Formulations

Therapeutic formulations of the anti-TAHO antibodies, TAHO bindingoligopeptides, TAHO binding organic molecules and/or TAHO polypeptidesused in accordance with the present invention are prepared for storageby mixing the antibody, polypeptide, oligopeptide or organic moleculehaving the desired degree of purity with optional pharmaceuticallyacceptable carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as acetate, Tris,phosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; tonicifiers such as trehaloseand sodium chloride; sugars such as sucrose, mannitol, trehalose orsorbitol; surfactant such as polysorbate; salt-forming counter-ions suchas sodium; metal complexes (e.g., Zn-protein complexes); and/ornon-ionic surfactants such as TWEEN®, PLURONICS® or polyethylene glycol(PEG). The antibody preferably comprises the antibody at a concentrationof between 5-200 mg/ml, preferably between 10-100 mg/ml.

The formulations herein may also contain more than one active compoundas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. For example, in addition to an anti-TAHO antibody, TAHO bindingoligopeptide, or TAHO binding organic molecule, it may be desirable toinclude in the one formulation, an additional antibody, e.g., a secondanti-TAHO antibody which binds a different epitope on the TAHOpolypeptide, or an antibody to some other target such as a growth factorthat affects the growth of the particular cancer. Alternatively, oradditionally, the composition may further comprise a chemotherapeuticagent, cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonalagent, and/or cardioprotectant. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT®(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

K. Treatment with Anti-TAHO Antibodies, TAHO Binding Oligopeptides andTAHO Binding Organic Molecules

To determine TAHO expression in the cancer, various detection assays areavailable. In one embodiment, TAHO polypeptide overexpression may beanalyzed by immunohistochemistry (IHC). Paffafin embedded tissuesections from a tumor biopsy may be subjected to the IHC assay andaccorded a TAHO protein staining intensity criteria as follows:

Score 0— no staining is observed or membrane staining is observed inless than 10% of tumor cells.

Score 1+—a faint/barely perceptible membrane staining is detected inmore than 10% of the tumor cells. The cells are only stained in part oftheir membrane.

Score 2+—a weak to moderate complete membrane staining is observed inmore than 10% of the tumor cells.

Score 3+—a moderate to strong complete membrane staining is observed inmore than 10% of the tumor cells.

Those tumors with 0 or 1+ scores for TAHO polypeptide expression may becharacterized as not overexpressing TAHO, whereas those tumors with 2+or 3+ scores may be characterized as overexpressing TAHO.

Alternatively, or additionally, FISH assays such as the INFORM® (sold byVentana, Ariz.) or PATHVISION® (Vysis, Ill.) may be carried out onformalin-fixed, paraffin-embedded tumor tissue to determine the extent(if any) of TAHO overexpression in the tumor.

TAHO overexpression or amplification may be evaluated using an in vivodetection assay, e.g., by administering a molecule (such as an antibody,oligopeptide or organic molecule) which binds the molecule to bedetected and is tagged with a detectable label (e.g., a radioactiveisotope or a fluorescent label) and externally scanning the patient forlocalization of the label.

As described above, the anti-TAHO antibodies, oligopeptides and organicmolecules of the invention have various non-therapeutic applications.The anti-TAHO antibodies, oligopeptides and organic molecules of thepresent invention can be useful for staging of TAHOpolypeptide-expressing cancers (e.g., in radioimaging). The antibodies,oligopeptides and organic molecules are also useful for purification orimmunoprecipitation of TAHO polypeptide from cells, for detection andquantitation of TAHO polypeptide in vitro, e.g., in an ELISA or aWestern blot, to kill and eliminate TAHO-expressing cells from apopulation of mixed cells as a step in the purification of other cells.

Currently, depending on the stage of the cancer, cancer treatmentinvolves one or a combination of the following therapies: surgery toremove the cancerous tissue, radiation therapy, and chemotherapy.Anti-TAHO antibody, oligopeptide or organic molecule therapy may beespecially desirable in elderly patients who do not tolerate thetoxicity and side effects of chemotherapy well and in metastatic diseasewhere radiation therapy has limited usefulness. The tumor targetinganti-TAHO antibodies, oligopeptides and organic molecules of theinvention are useful to alleviate TAHO-expressing cancers upon initialdiagnosis of the disease or during relapse. For therapeuticapplications, the anti-TAHO antibody, oligopeptide or organic moleculecan be used alone, or in combination therapy with, e.g., hormones,antiangiogens, or radiolabelled compounds, or with surgery, cryotherapy,and/or radiotherapy. Anti-TAHO antibody, oligopeptide or organicmolecule treatment can be administered in conjunction with other formsof conventional therapy, either consecutively with, pre- orpost-conventional therapy. Chemotherapeutic drugs such as TAXOTERE®(docetaxel), TAXOL® (palictaxel), estramustine and mitoxantrone are usedin treating cancer, in particular, in good risk patients. In the presentmethod of the invention for treating or alleviating cancer, the cancerpatient can be administered anti-TAHO antibody, oligopeptide or organicmolecule in conjunction with treatment with the one or more of thepreceding chemotherapeutic agents. In particular, combination therapywith palictaxel and modified derivatives (see, e.g., EP0600517) iscontemplated. The anti-TAHO antibody, oligopeptide or organic moleculewill be administered with a therapeutically effective dose of thechemotherapeutic agent. In another embodiment, the anti-TAHO antibody,oligopeptide or organic molecule is administered in conjunction withchemotherapy to enhance the activity and efficacy of thechemotherapeutic agent, e.g., paclitaxel. The Physicians' Desk Reference(PDR) discloses dosages of these agents that have been used in treatmentof various cancers. The dosing regimen and dosages of theseaforementioned chemotherapeutic drugs that are therapeutically effectivewill depend on the particular cancer being treated, the extent of thedisease and other factors familiar to the physician of skill in the artand can be determined by the physician.

In one particular embodiment, a conjugate comprising an anti-TAHOantibody, oligopeptide or organic molecule conjugated with a cytotoxicagent is administered to the patient. Preferably, the immunoconjugatebound to the TAHO protein is internalized by the cell, resulting inincreased therapeutic efficacy of the immunoconjugate in killing thecancer cell to which it binds. In a preferred embodiment, the cytotoxicagent targets or interferes with the nucleic acid in the cancer cell.Examples of such cytotoxic agents are described above and includemaytansinoids, calicheamicins, ribonucleases and DNA endonucleases.

The anti-TAHO antibodies, oligopeptides, organic molecules or toxinconjugates thereof are administered to a human patient, in accord withknown methods, such as intravenous administration, e.g., as a bolus orby continuous infusion over a period of time, by intramuscular,intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,intrasynovial, intrathecal, oral, topical, or inhalation routes.Intravenous or subcutaneous administration of the antibody, oligopeptideor organic molecule is preferred.

Other therapeutic regimens may be combined with the administration ofthe anti-TAHO antibody, oligopeptide or organic molecule. The combinedadministration includes co-administration, using separate formulationsor a single pharmaceutical formulation, and consecutive administrationin either order, wherein preferably there is a time period while both(or all) active agents simultaneously exert their biological activities.Preferably such combined therapy results in a synergistic therapeuticeffect.

It may also be desirable to combine administration of the anti-TAHOantibody or antibodies, oligopeptides or organic molecules, withadministration of an antibody directed against another tumor antigenassociated with the particular cancer.

In another embodiment, the therapeutic treatment methods of the presentinvention involves the combined administration of an anti-TAHO antibody(or antibodies), oligopeptides or organic molecules and one or morechemotherapeutic agents or growth inhibitory agents, includingco-administration of cocktails of different chemotherapeutic agents.Chemotherapeutic agents include estramustine phosphate, prednimustine,cisplatin, 5-fluorouracil, melphalan, cyclophosphamide, hydroxyurea andhydroxyureataxanes (such as paclitaxel and doxetaxel) and/oranthracycline antibiotics. Preparation and dosing schedules for suchchemotherapeutic agents may be used according to manufacturers'instructions or as determined empirically by the skilled practitioner.Preparation and dosing schedules for such chemotherapy are alsodescribed in Chemotherapy Service Ed., M. C. Perry, Williams & Wilkins,Baltimore, Md. (1992).

The antibody, oligopeptide or organic molecule may be combined with ananti-hormonal compound; e.g., an anti-estrogen compound such astamoxifen; an anti-progesterone such as onapristone (see, EP 616 812);or an anti-androgen such as flutamide, in dosages known for suchmolecules. Where the cancer to be treated is androgen independentcancer, the patient may previously have been subjected to anti-androgentherapy and, after the cancer becomes androgen independent, theanti-TAHO antibody, oligopeptide or organic molecule (and optionallyother agents as described herein) may be administered to the patient.

Sometimes, it may be beneficial to also co-administer a cardioprotectant(to prevent or reduce myocardial dysfunction associated with thetherapy) or one or more cytokines to the patient. In addition to theabove therapeutic regimes, the patient may be subjected to surgicalremoval of cancer cells and/or radiation therapy, before, simultaneouslywith, or post antibody, oligopeptide or organic molecule therapy.Suitable dosages for any of the above co-administered agents are thosepresently used and may be lowered due to the combined action (synergy)of the agent and anti-TAHO antibody, oligopeptide or organic molecule.

For the prevention or treatment of disease, the dosage and mode ofadministration will be chosen by the physician according to knowncriteria. The appropriate dosage of antibody, oligopeptide or organicmolecule will depend on the type of disease to be treated, as definedabove, the severity and course of the disease, whether the antibody,oligopeptide or organic molecule is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, oligopeptide or organic molecule, and thediscretion of the attending physician. The antibody, oligopeptide ororganic molecule is suitably administered to the patient at one time orover a series of treatments. Preferably, the antibody, oligopeptide ororganic molecule is administered by intravenous infusion or bysubcutaneous injections. Depending on the type and severity of thedisease, about 1 μg/kg to about 50 mg/kg body weight (e.g., about 0.1-15mg/kg/dose) of antibody can be an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A dosing regimencan comprise administering an initial loading dose of about 4 mg/kg,followed by a weekly maintenance dose of about 2 mg/kg of the anti-TAHOantibody. However, other dosage regimens may be useful. A typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. For repeated administrations over severaldays or longer, depending on the condition, the treatment is sustaineduntil a desired suppression of disease symptoms occurs. The progress ofthis therapy can be readily monitored by conventional methods and assaysand based on criteria known to the physician or other persons of skillin the art.

Aside from administration of the antibody protein to the patient, thepresent application contemplates administration of the antibody by genetherapy. Such administration of nucleic acid encoding the antibody isencompassed by the expression “administering a therapeutically effectiveamount of an antibody”. See, for example, WO96/07321 published Mar. 14,1996 concerning the use of gene therapy to generate intracellularantibodies.

There are two major approaches to getting the nucleic acid (optionallycontained in a vector) into the patient's cells; in vivo and ex vivo.For in vivo delivery the nucleic acid is injected directly into thepatient, usually at the site where the antibody is required. For ex vivotreatment, the patient's cells are removed, the nucleic acid isintroduced into these isolated cells and the modified cells areadministered to the patient either directly or, for example,encapsulated within porous membranes which are implanted into thepatient (see, e.g., U.S. Pat. Nos. 4,892,538 and 5,283,187). There are avariety of techniques available for introducing nucleic acids intoviable cells. The techniques vary depending upon whether the nucleicacid is transferred into cultured cells in vitro, or in vivo in thecells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. A commonly used vector for ex vivodelivery of the gene is a retroviral vector.

The currently preferred in vivo nucleic acid transfer techniques includetransfection with viral vectors (such as adenovirus, Herpes simplex Ivirus, or adeno-associated virus) and lipid-based systems (useful lipidsfor lipid-mediated transfer of the gene are DOTMA, DOPE and DC-Chol, forexample). For review of the currently known gene marking and genetherapy protocols see Anderson et al., Science 256:808-813 (1992). Seealso WO 93/25673 and the references cited therein.

The anti-TAHO antibodies of the invention can be in the different formsencompassed by the definition of “antibody” herein. Thus, the antibodiesinclude full length or intact antibody, antibody fragments, nativesequence antibody or amino acid variants, humanized, chimeric or fusionantibodies, immunoconjugates, and functional fragments thereof. Infusion antibodies an antibody sequence is fused to a heterologouspolypeptide sequence. The antibodies can be modified in the Fc region toprovide desired effector functions. As discussed in more detail in thesections herein, with the appropriate Fc regions, the naked antibodybound on the cell surface can induce cytotoxicity, e.g., viaantibody-dependent cellular cytotoxicity (ADCC) or by recruitingcomplement in complement dependent cytotoxicity, or some othermechanism. Alternatively, where it is desirable to eliminate or reduceeffector function, so as to minimize side effects or therapeuticcomplications, certain other Fc regions may be used.

In one embodiment, the antibody competes for binding or bindsubstantially to, the same epitope as the antibodies of the invention.Antibodies having the biological characteristics of the presentanti-TAHO antibodies of the invention are also contemplated,specifically including the in vivo tumor targeting and any cellproliferation inhibition or cytotoxic characteristics.

Methods of producing the above antibodies are described in detailherein.

The present anti-TAHO antibodies, oligopeptides and organic moleculesare useful for treating a TAHO-expressing cancer or alleviating one ormore symptoms of the cancer in a mammal. Such a cancer includes, but isnot limited to, hematopoietic cancers or blood-related cancers, such aslymphoma, leukemia, myeloma or lymphoid malignancies, but also cancersof the spleen and cancers of the lymph nodes. More particular examplesof such B-cell associated cancers, including for example, high,intermediate and low grade lymphomas (including B cell lymphomas suchas, for example, mucosa-associated-lymphoid tissue B cell lymphoma andnon-Hodgkin's lymphoma, mantle cell lymphoma, Burkitt's lymphoma, smalllymphocytic lymphoma, marginal zone lymphoma, diffuse large celllymphoma, follicular lymphoma, and Hodgkin's lymphoma and T celllymphomas) and leukemias (including secondary leukemia, chroniclymphocytic leukemia, such as B cell leukemia (CD5+B lymphocytes),myeloid leukemia, such as acute myeloid leukemia, chronic myeloidleukemia, lymphoid leukemia, such as acute lymphoblastic leukemia andmyelodysplasia), multiple myeloma, such as plasma cell malignancy, andother hematological and/or B cell- or T-cell-associated cancers. Thecancers encompass metastatic cancers of any of the preceding. Theantibody, oligopeptide or organic molecule is able to bind to at least aportion of the cancer cells that express TAHO polypeptide in the mammal.In a preferred embodiment, the antibody, oligopeptide or organicmolecule is effective to destroy or kill TAHO-expressing tumor cells orinhibit the growth of such tumor cells, in vitro or in vivo, uponbinding to TAHO polypeptide on the cell. Such an antibody includes anaked anti-TAHO antibody (not conjugated to any agent). Naked antibodiesthat have cytotoxic or cell growth inhibition properties can be furtherharnessed with a cytotoxic agent to render them even more potent intumor cell destruction. Cytotoxic properties can be conferred to ananti-TAHO antibody by, e.g., conjugating the antibody with a cytotoxicagent, to form an immunoconjugate as described herein. The cytotoxicagent or a growth inhibitory agent is preferably a small molecule.Toxins such as calicheamicin or a maytansinoid and analogs orderivatives thereof, are preferable.

The invention provides a composition comprising an anti-TAHO antibody,oligopeptide or organic molecule of the invention, and a carrier. Forthe purposes of treating cancer, compositions can be administered to thepatient in need of such treatment, wherein the composition can compriseone or more anti-TAHO antibodies present as an immunoconjugate or as thenaked antibody. In a further embodiment, the compositions can comprisethese antibodies, oligopeptides or organic molecules in combination withother therapeutic agents such as cytotoxic or growth inhibitory agents,including chemotherapeutic agents. The invention also providesformulations comprising an anti-TAHO antibody, oligopeptide or organicmolecule of the invention, and a carrier. In one embodiment, theformulation is a therapeutic formulation comprising a pharmaceuticallyacceptable carrier.

Another aspect of the invention is isolated nucleic acids encoding theanti-TAHO antibodies. Nucleic acids encoding both the H and L chains andespecially the hypervariable region residues, chains which encode thenative sequence antibody as well as variants, modifications andhumanized versions of the antibody, are encompassed.

The invention also provides methods useful for treating a TAHOpolypeptide-expressing cancer or alleviating one or more symptoms of thecancer in a mammal, comprising administering a therapeutically effectiveamount of an anti-TAHO antibody, oligopeptide or organic molecule to themammal. The antibody, oligopeptide or organic molecule therapeuticcompositions can be administered short term (acute) or chronic, orintermittent as directed by physician. Also provided are methods ofinhibiting the growth of, and killing a TAHO polypeptide-expressingcell.

The invention also provides kits and articles of manufacture comprisingat least one anti-TAHO antibody, oligopeptide or organic molecule. Kitscontaining anti-TAHO antibodies, oligopeptides or organic molecules finduse, e.g., for TAHO cell killing assays, for purification orimmunoprecipitation of TAHO polypeptide from cells. For example, forisolation and purification of TAHO, the kit can contain an anti-TAHOantibody, oligopeptide or organic molecule coupled to beads (e.g.,sepharose beads). Kits can be provided which contain the antibodies,oligopeptides or organic molecules for detection and quantitation ofTAHO in vitro, e.g., in an ELISA or a Western blot. Such antibody,oligopeptide or organic molecule useful for detection may be providedwith a label such as a fluorescent or radiolabel.

L. Articles of Manufacture and Kits

Another embodiment of the invention is an article of manufacturecontaining materials useful for the treatment of anti-TAHO expressingcancer. The article of manufacture comprises a container and a label orpackage insert on or associated with the container. Suitable containersinclude, for example, bottles, vials, syringes, etc. The containers maybe formed from a variety of materials such as glass or plastic. Thecontainer holds a composition which is effective for treating the cancercondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an anti-TAHO antibody, oligopeptide or organic moleculeof the invention. The label or package insert indicates that thecomposition is used for treating cancer. The label or package insertwill further comprise instructions for administering the antibody,oligopeptide or organic molecule composition to the cancer patient.Additionally, the article of manufacture may further comprise a secondcontainer comprising a pharmaceutically-acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes.

Kits are also provided that are useful for various purposes, e.g., forTAHO-expressing cell killing assays, for purification orimmunoprecipitation of TAHO polypeptide from cells. For isolation andpurification of TAHO polypeptide, the kit can contain an anti-TAHOantibody, oligopeptide or organic molecule coupled to beads (e.g.,sepharose beads). Kits can be provided which contain the antibodies,oligopeptides or organic molecules for detection and quantitation ofTAHO polypeptide in vitro, e.g., in an ELISA or a Western blot. As withthe article of manufacture, the kit comprises a container and a label orpackage insert on or associated with the container. The container holdsa composition comprising at least one anti-TAHO antibody, oligopeptideor organic molecule of the invention. Additional containers may beincluded that contain, e.g., diluents and buffers, control antibodies.The label or package insert may provide a description of the compositionas well as instructions for the intended in vitro or detection use.

M. Uses for TAHO Polypeptides and TAHO-Polypeptide Encoding NucleicAcids

Nucleotide sequences (or their complement) encoding TAHO polypeptideshave various applications in the art of molecular biology, includinguses as hybridization probes, in chromosome and gene mapping and in thegeneration of anti-sense RNA and DNA probes. TAHO-encoding nucleic acidwill also be useful for the preparation of TAHO polypeptides by therecombinant techniques described herein, wherein those TAHO polypeptidesmay find use, for example, in the preparation of anti-TAHO antibodies asdescribed herein.

The full-length native sequence TAHO gene, or portions thereof, may beused as hybridization probes for a cDNA library to isolate thefull-length TAHO cDNA or to isolate still other cDNAs (for instance,those encoding naturally-occurring variants of TAHO or TAHO from otherspecies) which have a desired sequence identity to the native TAHOsequence disclosed herein. Optionally, the length of the probes will beabout 20 to about 50 bases. The hybridization probes may be derived fromat least partially novel regions of the full length native nucleotidesequence wherein those regions may be determined without undueexperimentation or from genomic sequences including promoters, enhancerelements and introns of native sequence TAHO. By way of example, ascreening method will comprise isolating the coding region of the TAHOgene using the known DNA sequence to synthesize a selected probe ofabout 40 bases. Hybridization probes may be labeled by a variety oflabels, including radionucleotides such as ³²P or ³⁵S, or enzymaticlabels such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems. Labeled probes having a sequencecomplementary to that of the TAHO gene of the present invention can beused to screen libraries of human cDNA, genomic DNA or mRNA to determinewhich members of such libraries the probe hybridizes to. Hybridizationtechniques are described in further detail in the Examples below. AnyEST sequences disclosed in the present application may similarly beemployed as probes, using the methods disclosed herein.

Other useful fragments of the TAHO-encoding nucleic acids includeantisense or sense oligonucleotides comprising a singe-stranded nucleicacid sequence (either RNA or DNA) capable of binding to target TAHO mRNA(sense) or TAHO DNA (antisense) sequences. Antisense or senseoligonucleotides, according to the present invention, comprise afragment of the coding region of TAHO DNA. Such a fragment generallycomprises at least about 14 nucleotides, preferably from about 14 to 30nucleotides. The ability to derive an antisense or a senseoligonucleotide, based upon a cDNA sequence encoding a given protein isdescribed in, for example, Stein and Cohen (Cancer Res. 48:2659, 1988)and van der Krol et al. (BioTechniques 6:958, 1988).

Binding of antisense or sense oligonucleotides to target nucleic acidsequences results in the formation of duplexes that block transcriptionor translation of the target sequence by one of several means, includingenhanced degradation of the duplexes, premature termination oftranscription or translation, or by other means. Such methods areencompassed by the present invention. The antisense oligonucleotidesthus may be used to block expression of TAHO proteins, wherein thoseTAHO proteins may play a role in the induction of cancer in mammals.Antisense or sense oligonucleotides further comprise oligonucleotideshaving modified sugar-phosphodiester backbones (or other sugar linkages,such as those described in WO 91/06629) and wherein such sugar linkagesare resistant to endogenous nucleases. Such oligonucleotides withresistant sugar linkages are stable in vivo (i.e., capable of resistingenzymatic degradation) but retain sequence specificity to be able tobind to target nucleotide sequences.

Preferred intragenic sites for antisense binding include the regionincorporating the translation initiation/start codon (5′-AUG/5′-ATG) ortermination/stop codon (5′-UAA, 5′-UAG and 5-UGA/5′-TAA, 5′-TAG and5′-TGA) of the open reading frame (ORF) of the gene. These regions referto a portion of the mRNA or gene that encompasses from about 25 to about50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation initiation or termination codon. Other preferred regions forantisense binding include: introns; exons; intron-exon junctions; theopen reading frame (ORF) or “coding region,” which is the region betweenthe translation initiation codon and the translation termination codon;the 5′ cap of an mRNA which comprises an N7-methylated guanosine residuejoined to the 5′-most residue of the mRNA via a 5′-5′ triphosphatelinkage and includes 5′ cap structure itself as well as the first 50nucleotides adjacent to the cap; the 5′ untranslated region (5′UTR), theportion of an mRNA in the 5′ direction from the translation initiationcodon, and thus including nucleotides between the 5′ cap site and thetranslation initiation codon of an mRNA or corresponding nucleotides onthe gene; and the 3′ untranslated region (3′UTR), the portion of an mRNAin the 3′ direction from the translation termination codon, and thusincluding nucleotides between the translation termination codon and 3′end of an mRNA or corresponding nucleotides on the gene.

Specific examples of preferred antisense compounds useful for inhibitingexpression of TAHO proteins include oligonucleotides containing modifiedbackbones or non-natural internucleoside linkages. Oligonucleotideshaving modified backbones include those that retain a phosphorus atom inthe backbone and those that do not have a phosphorus atom in thebackbone. For the purposes of this specification, and as sometimesreferenced in the art, modified oligonucleotides that do not have aphosphorus atom in their internucleoside backbone can also be consideredto be oligonucleosides. Preferred modified oligonucleotide backbonesinclude, for example, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotri-esters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates and chiral phosphonates, phosphinates,phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand borano-phosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Preferred oligonucleotides having inverted polarity comprise a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e. a singleinverted nucleoside residue which may be abasic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included. Representative UnitedStates patents that teach the preparation of phosphorus-containinglinkages include, but are not limited to, U.S. Pat. Nos. 3,687,808;4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423;5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939;5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821;5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599;5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, each of whichis herein incorporated by reference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH.sub.2 component parts. RepresentativeUnited States patents that teach the preparation of sucholigonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, each of whichis herein incorporated by reference.

In other preferred antisense oligonucleotides, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

Preferred antisense oligonucleotides incorporate phosphorothioatebackbones and/or heteroatom backbones, and in particular —CH₂—NH—O—CH₂—,—CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone],—CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂—[wherein the native phosphodiester backbone is represented as—O—P—O—CH₂—] described in the above referenced U.S. Pat. No. 5,489,677,and the amide backbones of the above referenced U.S. Pat. No. 5,602,240.Also preferred are antisense oligonucleotides having morpholino backbonestructures of the above-referenced U.S. Pat. No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O-alkyl, S-alkyl, or N-alkyl; O-alkenyl,S-alkeynyl, or N-alkenyl; O-alkynyl, S-alkynyl or N-alkynyl; orO-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may besubstituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl andalkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred antisense oligonucleotides comprise one of the followingat the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl,alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃,OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.A preferred modification includes 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim.Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)—

A further preferred modification includes Locked Nucleic Acids (LNAs) inwhich the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of thesugar ring thereby forming a bicyclic sugar moiety. The linkage ispreferably a methelyne (—CH₂—)_(n) group bridging the 2′ oxygen atom andthe 4′ carbon atom wherein n is 1 or 2. LNAs and preparation thereof aredescribed in WO 98/39352 and WO 99/14226.

Other preferred modifications include 2′-methoxy(2′-O—CH₃),2′-aminopropoxy(2′-OCH₂CH₂CH₂ NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, each of which is herein incorporated byreference in its entirety.

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃ or —CH₂—C≡CH) uracil andcytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil,8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine,8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and3-deazaguanine and 3-deazaadenine. Further modified nucleobases includetricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, and thosedisclosed by Englisch et al., Angewandte Chemie, International Edition,1991, 30, 613. Certain of these nucleobases are particularly useful forincreasing the binding affinity of the oligomeric compounds of theinvention. These include 5-substituted pyrimidines, 6-azapyrimidines andN-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutionshave been shown to increase nucleic acid duplex stability by0.6-1.2.degree. C. (Sanghvi et al, Antisense Research and Applications,CRC Press, Boca Raton, 1993, pp. 276-278) and are preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications. Representative United Statespatents that teach the preparation of modified nucleobases include, butare not limited to: U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272;5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540;5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653;5,763,588; 6,005,096; 5,681,941 and 5,750,692, each of which is hereinincorporated by reference.

Another modification of antisense oligonucleotides chemically linking tothe oligonucleotide one or more moieties or conjugates which enhance theactivity, cellular distribution or cellular uptake of theoligonucleotide. The compounds of the invention can include conjugategroups covalently bound to functional groups such as primary orsecondary hydroxyl groups. Conjugate groups of the invention includeintercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, polyethers, groups that enhance the pharmacodynamic propertiesof oligomers, and groups that enhance the pharmacokinetic properties ofoligomers. Typical conjugates groups include cholesterols, lipids,cation lipids, phospholipids, cationic phospholipids, biotin, phenazine,folate, phenanthridine, anthraquinone, acridine, fluoresceins,rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamicproperties, in the context of this invention, include groups thatimprove oligomer uptake, enhance oligomer resistance to degradation,and/or strengthen sequence-specific hybridization with RNA. Groups thatenhance the pharmacokinetic properties, in the context of thisinvention, include groups that improve oligomer uptake, distribution,metabolism or excretion. Conjugate moieties include but are not limitedto lipid moieties such as a cholesterol moiety (Letsinger et al., Proc.Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan etal., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g.,hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660,306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770),a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20,533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues(Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al.,FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75,49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety. Oligonucleotides of theinvention may also be conjugated to active drug substances, for example,aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen,ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine,2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, abenzothiadiazide, chlorothiazide, a diazepine, indomethicin, abarbiturate, a cephalosporin, a sulfa drug, an antidiabetic, anantibacterial or an antibiotic. Oligonucleotide-drug conjugates andtheir preparation are described in U.S. patent application Ser. No.09/334,130 (filed Jun. 15, 1999) and U.S. Pat. Nos. 4,828,979;4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538;5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802;5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046;4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941;4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963;5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469;5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241,5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785;5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726;5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of which is hereinincorporated by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds which are chimeric compounds. “Chimeric”antisense compounds or “chimeras,” in the context of this invention, areantisense compounds, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the oligonucleotide may serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase H is a cellular endonuclease which cleaves the RNA strandof an RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide inhibition of gene expression. Consequently, comparableresults can often be obtained with shorter oligonucleotides whenchimeric oligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Chimericantisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Preferred chimeric antisense oligonucleotides incorporate at least one2′ modified sugar (preferably 2′-O—(CH₂)₂—O—CH₃) at the 3′ terminal toconfer nuclease resistance and a region with at least 4 contiguous 2′-Hsugars to confer RNase H activity. Such compounds have also beenreferred to in the art as hybrids or gapmers. Preferred gapmers have aregion of 2′ modified sugars (preferably 2′-O—(CH₂)₂—O—CH₃) at the3′-terminal and at the 5′ terminal separated by at least one regionhaving at least 4 contiguous 2′-H sugars and preferably incorporatephosphorothioate backbone linkages. Representative United States patentsthat teach the preparation of such hybrid structures include, but arenot limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007;5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065;5,652,355; 5,652,356; and 5,700,922, each of which is hereinincorporated by reference in its entirety.

The antisense compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives. The compounds of theinvention may also be admixed, encapsulated, conjugated or otherwiseassociated with other molecules, molecule structures or mixtures ofcompounds, as for example, liposomes, receptor targeted molecules, oral,rectal, topical or other formulations, for assisting in uptake,distribution and/or absorption. Representative United States patentsthat teach the preparation of such uptake, distribution and/orabsorption assisting formulations include, but are not limited to, U.S.Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291;5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899;5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633;5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295;5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756,each of which is herein incorporated by reference.

Other examples of sense or antisense oligonucleotides include thoseoligonucleotides which are covalently linked to organic moieties, suchas those described in WO 90/10048, and other moieties that increasesaffinity of the oligonucleotide for a target nucleic acid sequence, suchas poly-(L-lysine). Further still, intercalating agents, such asellipticine, and alkylating agents or metal complexes may be attached tosense or antisense oligonucleotides to modify binding specificities ofthe antisense or sense oligonucleotide for the target nucleotidesequence.

Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. In a preferred procedure, an antisense or sense oligonucleotideis inserted into a suitable retroviral vector. A cell containing thetarget nucleic acid sequence is contacted with the recombinantretroviral vector, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, those derived from the murineretrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the doublecopy vectors designated DCT5A, DCT5B and DCT5C (see WO 90/13641).

Sense or antisense oligonucleotides also may be introduced into a cellcontaining the target nucleotide sequence by formation of a conjugatewith a ligand binding molecule, as described in WO 91/04753. Suitableligand binding molecules include, but are not limited to, cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors. Preferably, conjugation of the ligand bindingmolecule does not substantially interfere with the ability of the ligandbinding molecule to bind to its corresponding molecule or receptor, orblock entry of the sense or antisense oligonucleotide or its conjugatedversion into the cell.

Alternatively, a sense or an antisense oligonucleotide may be introducedinto a cell containing the target nucleic acid sequence by formation ofan oligonucleotide-lipid complex, as described in WO 90/10448. The senseor antisense oligonucleotide-lipid complex is preferably dissociatedwithin the cell by an endogenous lipase.

Antisense or sense RNA or DNA molecules are generally at least about 5nucleotides in length, alternatively at least about 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110,115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180,185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440,450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580,590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720,730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860,870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000nucleotides in length, wherein in this context the term “about” meansthe referenced nucleotide sequence length plus or minus 10% of thatreferenced length.

The probes may also be employed in PCR techniques to generate a pool ofsequences for identification of closely related TAHO coding sequences.

Nucleotide sequences encoding a TAHO can also be used to constructhybridization probes for mapping the gene which encodes that TAHO andfor the genetic analysis of individuals with genetic disorders. Thenucleotide sequences provided herein may be mapped to a chromosome andspecific regions of a chromosome using known techniques, such as in situhybridization, linkage analysis against known chromosomal markers, andhybridization screening with libraries.

When the coding sequences for TAHO encode a protein which binds toanother protein (example, where the TAHO is a receptor), the TAHO can beused in assays to identify the other proteins or molecules involved inthe binding interaction. By such methods, inhibitors of thereceptor/ligand binding interaction can be identified. Proteins involvedin such binding interactions can also be used to screen for peptide orsmall molecule inhibitors or agonists of the binding interaction. Also,the receptor TAHO can be used to isolate correlative ligand(s).Screening assays can be designed to find lead compounds that mimic thebiological activity of a native TAHO or a receptor for TAHO. Suchscreening assays will include assays amenable to high-throughputscreening of chemical libraries, making them particularly suitable foridentifying small molecule drug candidates. Small molecules contemplatedinclude synthetic organic or inorganic compounds. The assays can beperformed in a variety of formats, including protein-protein bindingassays, biochemical screening assays, immunoassays and cell basedassays, which are well characterized in the art.

Nucleic acids which encode TAHO or its modified forms can also be usedto generate either transgenic animals or “knock out” animals which, inturn, are useful in the development and screening of therapeuticallyuseful reagents. A transgenic animal (e.g., a mouse or rat) is an animalhaving cells that contain a transgene, which transgene was introducedinto the animal or an ancestor of the animal at a prenatal, e.g., anembryonic stage. A transgene is a DNA which is integrated into thegenome of a cell from which a transgenic animal develops. In oneembodiment, cDNA encoding TAHO can be used to clone genomic DNA encodingTAHO in accordance with established techniques and the genomic sequencesused to generate transgenic animals that contain cells which express DNAencoding TAHO. Methods for generating transgenic animals, particularlyanimals such as mice or rats, have become conventional in the art andare described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009.Typically, particular cells would be targeted for TAHO transgeneincorporation with tissue-specific enhancers. Transgenic animals thatinclude a copy of a transgene encoding TAHO introduced into the germline of the animal at an embryonic stage can be used to examine theeffect of increased expression of DNA encoding TAHO. Such animals can beused as tester animals for reagents thought to confer protection from,for example, pathological conditions associated with its overexpression.In accordance with this facet of the invention, an animal is treatedwith the reagent and a reduced incidence of the pathological condition,compared to untreated animals bearing the transgene, would indicate apotential therapeutic intervention for the pathological condition.

Alternatively, non-human homologues of TAHO can be used to construct aTAHO “knock out” animal which has a defective or altered gene encodingTAHO as a result of homologous recombination between the endogenous geneencoding TAHO and altered genomic DNA encoding TAHO introduced into anembryonic stem cell of the animal. For example, cDNA encoding TAHO canbe used to clone genomic DNA encoding TAHO in accordance withestablished techniques. A portion of the genomic DNA encoding TAHO canbe deleted or replaced with another gene, such as a gene encoding aselectable marker which can be used to monitor integration. Typically,several kilobases of unaltered flanking DNA (both at the 5′ and 3′ ends)are included in the vector [see e.g., Thomas and Capecchi, Cell, 51:503(1987) for a description of homologous recombination vectors]. Thevector is introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced DNA has homologouslyrecombined with the endogenous DNA are selected [see e.g., Li et al.,Cell, 69:915 (1992)]. The selected cells are then injected into ablastocyst of an animal (e.g., a mouse or rat) to form aggregationchimeras [see e.g., Bradley, in Teratocarcinomas and Embryonic StemCells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987),pp. 113-[52]. A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term tocreate a “knock out” animal. Progeny harboring the homologouslyrecombined DNA in their germ cells can be identified by standardtechniques and used to breed animals in which all cells of the animalcontain the homologously recombined DNA. Knockout animals can becharacterized for instance, for their ability to defend against certainpathological conditions and for their development of pathologicalconditions due to absence of the TAHO polypeptide.

Nucleic acid encoding the TAHO polypeptides may also be used in genetherapy. In gene therapy applications, genes are introduced into cellsin order to achieve in vivo synthesis of a therapeutically effectivegenetic product, for example for replacement of a defective gene. “Genetherapy” includes both conventional gene therapy where a lasting effectis achieved by a single treatment, and the administration of genetherapeutic agents, which involves the one time or repeatedadministration of a therapeutically effective DNA or mRNA. AntisenseRNAs and DNAs can be used as therapeutic agents for blocking theexpression of certain genes in vivo. It has already been shown thatshort antisense oligonucleotides can be imported into cells where theyact as inhibitors, despite their low intracellular concentrations causedby their restricted uptake by the cell membrane. (Zameenik et al., Proc.Natl. Acad. Sci. USA 83:4143-4146 [1986]). The oligonucleotides can bemodified to enhance their uptake, e.g. by substituting their negativelycharged phosphodiester groups by uncharged groups.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. The currently preferred in vivogene transfer techniques include transfection with viral (typicallyretroviral) vectors and viral coat protein-liposome mediatedtransfection (Dzau et al., Trends in Biotechnology 11, 205-210 [1993]).In some situations it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA 87, 3410-3414 (1990). For review of gene marking and genetherapy protocols see Anderson et al., Science 256, 808-813 (1992).

The nucleic acid molecules encoding the TAHO polypeptides or fragmentsthereof described herein are useful for chromosome identification. Inthis regard, there exists an ongoing need to identify new chromosomemarkers, since relatively few chromosome marking reagents, based uponactual sequence data are presently available. Each TAHO nucleic acidmolecule of the present invention can be used as a chromosome marker.

The TAHO polypeptides and nucleic acid molecules of the presentinvention may also be used diagnostically for tissue typing, wherein theTAHO polypeptides of the present invention may be differentiallyexpressed in one tissue as compared to another, preferably in a diseasedtissue as compared to a normal tissue of the same tissue type. TAHOnucleic acid molecules will find use for generating probes for PCR,Northern analysis, Southern analysis and Western analysis.

This invention encompasses methods of screening compounds to identifythose that mimic the TAHO polypeptide (agonists) or prevent the effectof the TAHO polypeptide (antagonists). Screening assays for antagonistdrug candidates are designed to identify compounds that bind or complexwith the TAHO polypeptides encoded by the genes identified herein, orotherwise interfere with the interaction of the encoded polypeptideswith other cellular proteins, including e.g., inhibiting the expressionof TAHO polypeptide from cells. Such screening assays will includeassays amenable to high-throughput screening of chemical libraries,making them particularly suitable for identifying small molecule drugcandidates.

The assays can be performed in a variety of formats, includingprotein-protein binding assays, biochemical screening assays,immunoassays, and cell-based assays, which are well characterized in theart.

All assays for antagonists are common in that they call for contactingthe drug candidate with a TAHO polypeptide encoded by a nucleic acididentified herein under conditions and for a time sufficient to allowthese two components to interact.

In binding assays, the interaction is binding and the complex formed canbe isolated or detected in the reaction mixture. In a particularembodiment, the TAHO polypeptide encoded by the gene identified hereinor the drug candidate is immobilized on a solid phase, e.g., on amicrotiter plate, by covalent or non-covalent attachments. Non-covalentattachment generally is accomplished by coating the solid surface with asolution of the TAHO polypeptide and drying. Alternatively, animmobilized antibody, e.g., a monoclonal antibody, specific for the TAHOpolypeptide to be immobilized can be used to anchor it to a solidsurface. The assay is performed by adding the non-immobilized component,which may be labeled by a detectable label, to the immobilizedcomponent, e.g., the coated surface containing the anchored component.When the reaction is complete, the non-reacted components are removed,e.g., by washing, and complexes anchored on the solid surface aredetected. When the originally non-immobilized component carries adetectable label, the detection of label immobilized on the surfaceindicates that complexing occurred. Where the originally non-immobilizedcomponent does not carry a label, complexing can be detected, forexample, by using a labeled antibody specifically binding theimmobilized complex.

If the candidate compound interacts with but does not bind to aparticular TAHO polypeptide encoded by a gene identified herein, itsinteraction with that polypeptide can be assayed by methods well knownfor detecting protein-protein interactions. Such assays includetraditional approaches, such as, e.g., cross-linking,co-immunoprecipitation, and co-purification through gradients orchromatographic columns. In addition, protein-protein interactions canbe monitored by using a yeast-based genetic system described by Fieldsand co-workers (Fields and Song, Nature (London), 340:245-246 (1989);Chien et al., Proc. Natl. Acad. Sci. USA, 88:9578-9582 (1991)) asdisclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89:5789-5793 (1991). Many transcriptional activators, such as yeast GAL4,consist of two physically discrete modular domains, one acting as theDNA-binding domain, the other one functioning as thetranscription-activation domain. The yeast expression system describedin the foregoing publications (generally referred to as the “two-hybridsystem”) takes advantage of this property, and employs two hybridproteins, one in which the target protein is fused to the DNA-bindingdomain of GAL4, and another, in which candidate activating proteins arefused to the activation domain. The expression of a GAL1-lacZ reportergene under control of a GAL4-activated promoter depends onreconstitution of GAL4 activity via protein-protein interaction.Colonies containing interacting polypeptides are detected with achromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™)for identifying protein-protein interactions between two specificproteins using the two-hybrid technique is commercially available fromClontech. This system can also be extended to map protein domainsinvolved in specific protein interactions as well as to pinpoint aminoacid residues that are crucial for these interactions.

Compounds that interfere with the interaction of a gene encoding a TAHOpolypeptide identified herein and other intra- or extracellularcomponents can be tested as follows: usually a reaction mixture isprepared containing the product of the gene and the intra- orextracellular component under conditions and for a time allowing for theinteraction and binding of the two products. To test the ability of acandidate compound to inhibit binding, the reaction is run in theabsence and in the presence of the test compound. In addition, a placebomay be added to a third reaction mixture, to serve as positive control.The binding (complex formation) between the test compound and the intra-or extracellular component present in the mixture is monitored asdescribed hereinabove. The formation of a complex in the controlreaction(s) but not in the reaction mixture containing the test compoundindicates that the test compound interferes with the interaction of thetest compound and its reaction partner.

To assay for antagonists, the TAHO polypeptide may be added to a cellalong with the compound to be screened for a particular activity and theability of the compound to inhibit the activity of interest in thepresence of the TAHO polypeptide indicates that the compound is anantagonist to the TAHO polypeptide. Alternatively, antagonists may bedetected by combining the TAHO polypeptide and a potential antagonistwith membrane-bound TAHO polypeptide receptors or recombinant receptorsunder appropriate conditions for a competitive inhibition assay. TheTAHO polypeptide can be labeled, such as by radioactivity, such that thenumber of TAHO polypeptide molecules bound to the receptor can be usedto determine the effectiveness of the potential antagonist. The geneencoding the receptor can be identified by numerous methods known tothose of skill in the art, for example, ligand panning and FACS sorting.Coligan et al., Current Protocols in Immun., 1(2): Chapter 5 (1991).Preferably, expression cloning is employed wherein polyadenylated RNA isprepared from a cell responsive to the TAHO polypeptide and a cDNAlibrary created from this RNA is divided into pools and used totransfect COS cells or other cells that are not responsive to the TAHOpolypeptide. Transfected cells that are grown on glass slides areexposed to labeled TAHO polypeptide. The TAHO polypeptide can be labeledby a variety of means including iodination or inclusion of a recognitionsite for a site-specific protein kinase. Following fixation andincubation, the slides are subjected to autoradiographic analysis.Positive pools are identified and sub-pools are prepared andre-transfected using an interactive sub-pooling and re-screeningprocess, eventually yielding a single clone that encodes the putativereceptor.

As an alternative approach for receptor identification, labeled TAHOpolypeptide can be photoaffinity-linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE and exposed to X-ray film. The labeled complexcontaining the receptor can be excised, resolved into peptide fragments,and subjected to protein micro-sequencing. The amino acid sequenceobtained from micro-sequencing would be used to design a set ofdegenerate oligonucleotide probes to screen a cDNA library to identifythe gene encoding the putative receptor.

In another assay for antagonists, mammalian cells or a membranepreparation expressing the receptor would be incubated with labeled TAHOpolypeptide in the presence of the candidate compound. The ability ofthe compound to enhance or block this interaction could then bemeasured.

More specific examples of potential antagonists include anoligonucleotide that binds to the fusions of immunoglobulin with TAHOpolypeptide, and, in particular, antibodies including, withoutlimitation, poly- and monoclonal antibodies and antibody fragments,single-chain antibodies, anti-idiotypic antibodies, and chimeric orhumanized versions of such antibodies or fragments, as well as humanantibodies and antibody fragments. Alternatively, a potential antagonistmay be a closely related protein, for example, a mutated form of theTAHO polypeptide that recognizes the receptor but imparts no effect,thereby competitively inhibiting the action of the TAHO polypeptide.

Another potential TAHO polypeptide antagonist is an antisense RNA or DNAconstruct prepared using antisense technology, where, e.g., an antisenseRNA or DNA molecule acts to block directly the translation of mRNA byhybridizing to targeted mRNA and preventing protein translation.Antisense technology can be used to control gene expression throughtriple-helix formation or antisense DNA or RNA, both of which methodsare based on binding of a polynucleotide to DNA or RNA. For example, the5′ coding portion of the polynucleotide sequence, which encodes themature TAHO polypeptides herein, is used to design an antisense RNAoligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix—see Lee et al., Nucl. AcidsRes., 6:3073 (1979); Cooney et al., Science, 241: 456 (1988); Dervan etal., Science, 251:1360 (1991)), thereby preventing transcription and theproduction of the TAHO polypeptide. The antisense RNA oligonucleotidehybridizes to the mRNA in vivo and blocks translation of the mRNAmolecule into the TAHO polypeptide (antisense—Okano, Neurochem., 56:560(1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression(CRC Press: Boca Raton, Fla., 1988). The oligonucleotides describedabove can also be delivered to cells such that the antisense RNA or DNAmay be expressed in vivo to inhibit production of the TAHO polypeptide.When antisense DNA is used, oligodeoxyribonucleotides derived from thetranslation-initiation site, e.g., between about −10 and +10 positionsof the target gene nucleotide sequence, are preferred.

Potential antagonists include small molecules that bind to the activesite, the receptor binding site, or growth factor or other relevantbinding site of the TAHO polypeptide, thereby blocking the normalbiological activity of the TAHO polypeptide. Examples of small moleculesinclude, but are not limited to, small peptides or peptide-likemolecules, preferably soluble peptides, and synthetic non-peptidylorganic or inorganic compounds.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. Ribozymes act by sequence-specific hybridization to thecomplementary target RNA, followed by endonucleolytic cleavage. Specificribozyme cleavage sites within a potential RNA target can be identifiedby known techniques. For further details see, e.g., Rossi, CurrentBiology, 4:469-471 (1994), and PCT publication No. WO 97/33551(published Sep. 18, 1997).

Nucleic acid molecules in triple-helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple-helix formation via Hoogsteenbase-pairing rules, which generally require sizeable stretches ofpurines or pyrimidines on one strand of a duplex. For further detailssee, e.g., PCT publication No. WO 97/33551, supra.

These small molecules can be identified by any one or more of thescreening assays discussed hereinabove and/or by any other screeningtechniques well known for those skilled in the art.

Isolated TAHO polypeptide-encoding nucleic acid can be used herein forrecombinantly producing TAHO polypeptide using techniques well known inthe art and as described herein. In turn, the produced TAHO polypeptidescan be employed for generating anti-TAHO antibodies using techniqueswell known in the art and as described herein.

Antibodies specifically binding a TAHO polypeptide identified herein, aswell as other molecules identified by the screening assays disclosedhereinbefore, can be administered for the treatment of variousdisorders, including cancer, in the form of pharmaceutical compositions.

If the TAHO polypeptide is intracellular and whole antibodies are usedas inhibitors, internalizing antibodies are preferred. However,lipofections or liposomes can also be used to deliver the antibody, oran antibody fragment, into cells. Where antibody fragments are used, thesmallest inhibitory fragment that specifically binds to the bindingdomain of the target protein is preferred. For example, based upon thevariable-region sequences of an antibody, peptide molecules can bedesigned that retain the ability to bind the target protein sequence.Such peptides can be synthesized chemically and/or produced byrecombinant DNA technology. See, e.g., Marasco et al., Proc. Natl. Acad.Sci. USA, 90: 7889-7893 (1993).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Alternatively, or in addition, the composition may comprise an agentthat enhances its function, such as, for example, a cytotoxic agent,cytokine, chemotherapeutic agent, or growth-inhibitory agent. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated.Antibodies used in the examples are commercially available antibodiesand include, but are not limited to, anti-CD180 (eBioscience MRH73-11,BD Pharmingen G28-8) and Serotec MHR73), anti-CD20 (Ancell 2H7 and BDPharmingen 2H7), anti-CD72 (BD Pharmingen J4-117), anti-CXCR5 (R&DSystems 51505), anti-CD22 (Ancell RFB4, DAKO To15, Diatec 157, SigmaHIB-22 and Monosan BL-BC34), anti-CD22 (Leinco RFB-4 and NeoMarkers22C04), anti-CD21 (ATCC HB-135, which is herein referred to also as ATCCHB5, THB-5 and HB-5), anti-HLA-DOB (BD Pharmingen DOB.L1), anti-CD79a(Caltag ZL7-4 and Serotec ZL7-4), anti-CD79b (Biomeda SN8 and BDPharmingen CB-3), anti-CD19 (Biomeda CB-19, also herein referred to asB496), anti-FCER2 (Ancell BU38 and Serotec D3.6 and BD PharmingenM-L233). The source of those cells identified in the following examples,and throughout the specification, by ATCC accession numbers is theAmerican Type Culture Collection, Manassas, Va.

Example 1 Microarray Data Analysis of TAHO Expression

Microarray data involves the analysis of TAHO expression by theperformance of DNA microarray analysis on a wide a variety of RNAsamples from tissues and cultured cells. Samples include normal andcancerous human tissue and various kinds of purified immune cells bothat rest and following external stimulation. These RNA samples may beanalyzed according to regular microarray protocols on Agilentmicroarrays.

In this experiment, RNA was isolated from cells and cyanine-3 andcyanine-5 labeled cRNA probes were generated by in vitro transcriptionusing the Agilent Low Input RNA Fluorescent Linear Amplification Kit(Agilent). Cyanine-5 was used to label the samples to be tested forexpression of the PRO polypeptide, for example, the myeloma and plasmacells, and cyanine-3 was used to label the universal reference (theStratagene cell line pool) with which the expression of the test sampleswere compared. 0.1 μg-0.2 μg of cyanine-3 and cyanine-5 labeled cRNAprobe was hybridized to Agilent 60-mer oligonucleotide array chips usingthe In Situ Hybridization Kit Plus (Agilent). These probes werehybridized to microarrays. For multiple myeloma analysis, probes werehybridized to Agilent Whole Human Genome oligonucleotide microarraysusing standard Agilent recommended conditions and buffers (Agilent).

The cRNA probes are hybridized to the microarrays at 60° C. for 17 hourson a hybridization rotator set at 4 RPM. After washing, the microarraysare scanned with the Agilent microarray scanner which is capable ofexciting and detecting the fluorescence from the cyanine-3 and cyanine-5fluorescent molecules (532 and 633 nm laser lines). The data for eachgene on the 60-mer oligonucleotide array was extracted from the scannedmicroarray image using Agilent feature extraction software whichaccounts for feature recognition, background subtraction andnormalization and the resulting data was loaded into the softwarepackage known as the Rosetta Resolver Gene Expression Data AnalysisSystem (Rosetta Inpharmatics, Inc.). Rosetta Resolver includes arelational database and numerous analytical tools to store, retrieve andanalyze large quantities of intensity or ratio gene expression data.

In this example, B cells and T cells (control) were obtained formicroarray analysis. For isolation of naive and memory B cells andplasma cells, human peripheral blood mononuclear cells (PBMC) wereseparated from either leukopack provided by four healthy male donors orfrom whole blood of several normal donors. CD138+ plasma cells wereisolated from PBMC using the MACS (Miltenyi Biotec) magnetic cellsorting system and anti-CD138 beads. Alternatively, total CD19+B cellswere selected with anti-CD19 beads and MACS sorting. After enrichment ofCD19+ (purity around 90%), FACS (Moflo) sorting was performed toseparate naive and memory B cells. Sorted cells were collected bysubjecting the samples to centrifugation. The sorted cells wereimmediately lysed in LTR buffer and homogenized with QIAshredder(Qiagen) spin column and followed by RNeasy mini kit for RNApurification. RNA yield was variable from 0.4-10 μg and depended on thecell numbers.

As a control, T cells were isolated for microarray analysis. Peripheralblood CD8 cells were isolated from leukopacks by negative selectionusing the Stem Cell Technologies CD8 cell isolation kit (RosetteSeparation) and further purified by the MACS magnetic cell sortingsystem using CD8 cell isolation kit and CD45RO microbeads were added toremove CD45RO cells (Miltenyi Biotec). CD8 T cells were divided into 3samples with each sample subjected to the stimulation as follows: (1)anti-CD3 and anti-CD28, plus IL-12 and anti-IL4 antibody, (2) anti-CD3and anti-CD29 without adding cytokines or neutralizing antibodies and(3) anti-CD3 and anti-CD28, plus IL-4, anti-IL12 antibody and anti-IFN-γantibody. 48 hours after stimulation, RNA was collected. After 72 hours,cells were expanded by adding diluting 8-fold with fresh media. 7 daysafter the RNA was collected, CD8 cells were collected, washed andrestimulated by anti-CD3 and anti-CD28. 16 hours later, a secondcollection of RNA was made. 48 hours after restimulation, a thirdcollection of RNA was made. RNA was collected by using Qiagen Midi prepsas per the instructions in the manual with the addition of an on-columnDNAse I digestion after the first RW1 wash step. RNA was eluted in RNAsefree water and subsequently concentrated by ethanol precipitation.Precipitated RNA was taken up in nuclease free water to a final minimumconcentration of 0.5 μg/μl.

Additional control microrrays were performed on RNA isolated from CD4+Thelper T cells, natural killer (NK) cells, neutrophils (N'phil), CD14+,CD16+ and CD16-monocytes and dendritic cells (DC).

Additional microarrays were performed on RNA isolated from canceroustissue, such as Non-Hodgkin's Lymphoma (NHL), follicular lymphoma (FL)and multiple myeloma (MM). Additional microarrays were performed on RNAisolated from normal cells, such as normal lymph node (NLN), normal Bcells, such as B cells from centroblasts, centrocytes and follicularmantel, memory B cells, and normal plasma cells (PC), which are from theB cell lineage and are normal counterparts of the myeloma cell, such astonsil plasma cells, bone marrow plasma cells (BM PC), CD19+ plasmacells (CD19+PC), CD19− plasma cells (CD19−PC). Additional microarrayswere performed on normal tissue, such as cerebellum, heart, prostate,adrenal, bladder, small intestine (s. intestine), colon, fetal liver,uterus, kidney, placenta, lung, pancreas, muscle, brain, salivary, bonemarrow (marrow), blood, thymus, tonsil, spleen, testes, and mammarygland.

The molecules listed below have been identified as being significantlyexpressed in B cells as compared to non-B cells. Specifically, themolecules are differentially expressed in naive B cells, memory B cellsthat are either IgGA+ or IgM+ and plasma cells from either PBMC or bonemarrow, in comparison to non-B cells, for example T cells. Accordingly,these molecules represent excellent targets for therapy of tumors inmammals. Molecule specific expression in: as compared to: DNA225875(TAHO6) B cells non-B cells DNA225820 (TAHO25) B cells non-B cellsSummary

In FIGS. 7-8, significant mRNA expression was generally indicated as aratio value of greater than 2 (vertical axis of FIGS. 7-8). In FIGS.7-8, any apparent expression in non-B cells, such as in prostate,spleen, etc. may represent an artifact, infiltration of normal tissue bylymphocytes or loss of sample integrity by the vendor.

(1) TAHO6 (also referred herein as CR2 and CD21) was significantlyexpressed in non-hodgkin's lymphoma (NHL) and normal lymph node (NLN).Further TAHO6 was significantly expressed in spleen FIG. 7).

(2) TAHO25 (also referred herein as CD19) was significantly expressed innon-hodgkin's lymphoma (NHL), normal lymph node (NLN), follicularlymphoma (FL), centroblasts, centrocytes, memory B cells and follicularmantle cells. Further TAHO25 was significantly expressed in tonsil andspleen (FIG. 8). However, as indicated above, any apparent expression innon-B cells, such as in prostate, spleen, blood, tonsil, etc. mayrepresent an artifact, infiltration of normal tissue by lymphocytes orloss of sample integrity by the vendor.

As CD21/TAHO6 and CD19/TAHO25 have been identified as beingsignificantly expressed in B cells and in samples from B-cell associateddiseases, such as Non-Hodgkin's lymphoma, follicular lymphoma andmultiple myeloma as compared to non-B cells as detected by microarrayanalysis, the molecules are excellent targets for therapy of tumors inmammals, including B-cell associated cancers, such as lymphomas,leukemias, myelomas and other cancers of hematopoietic cells.

Example 2 Quantitative Analysis of TAHO mRNA Expression

In this assay, a 5′ nuclease assay (for example, TaqMan®) and real-timequantitative PCR (for example, Mx3000P™ Real-Time PCR System(Stratagene, La Jolla, Calif.)), were used to find genes that aresignificantly overexpressed in a specific tissue type, such as B cells,as compared to a different cell type, such as other primary white bloodcell types, and which further may be overexpressed in cancerous cells ofthe specific tissue type as compared to non-cancerous cells of thespecific tissue type. The 5′ nuclease assay reaction is a fluorescentPCR-based technique which makes use of the 5′ exonuclease activity ofTaq DNA polymerase enzyme to monitor gene expression in real time. Twooligonucleotide primers (whose sequences are based upon the gene or ESTsequence of interest) are used to generate an amplicon typical of a PCRreaction. A third oligonucleotide, or probe, is designed to detectnucleotide sequence located between the two PCR primers. The probe isnon-extendible by Taq DNA polymerase enzyme, and is labeled with areporter fluorescent dye and a quencher fluorescent dye. Anylaser-induced emission from the reporter dye is quenched by thequenching dye when the two dyes are located close together as they areon the probe. During the PCR amplification reaction, the Taq DNApolymerase enzyme cleaves the probe in a template-dependent manner. Theresultant probe fragments disassociate in solution, and signal from thereleased reporter dye is free from the quenching effect of the secondfluorophore. One molecule of reporter dye is liberated for each newmolecule synthesized, and detection of the unquenched reporter dyeprovides the basis for quantitative interpretation of the data.

The 5′ nuclease procedure is run on a real-time quantitative PCR devicesuch as the Mx3000™ Real-Time PCR System. The system consists of athermocycler, a quartz-tungsten lamp, a photomultiplier tube (PMT) fordetection and a computer. The system amplifies samples in a 96-wellformat on a thermocycler.

During amplification, laser-induced fluorescent signal is collected inreal-time through fiber optics cables for all 96 wells, and detected atthe PMT. The system includes software for running the instrument and foranalyzing the data.

The starting material for the screen was mRNA (50 ng/well run induplicate) isolated from a variety of different white blood cell types(Neturophil (Neutr), Natural Killer cells (NK), Dendritic cells (Dend.),Monocytes (Mono), T cells (CD4+ and CD8+ subsets), stem cells (CD34+) aswell as 20 separate B cell donors (donor Ids 310, 330, 357, 362, 597,635, 816, 1012, 1013, 1020, 1072, 1074, 1075, 1076, 1077, 1086, 1096,1098, 1109, 1112) to test for donor variability. All RNA was purchasedcommercially (AllCells, LLC, Berkeley, Calif.) and the concentration ofeach was measured precisely upon receipt. The mRNA is quantitatedprecisely, e.g., fluorometrically.

5′ nuclease assay data are initially expressed as Ct, or the thresholdcycle. This is defined as the cycle at which the reporter signalaccumulates above the background level of fluorescence. The ΔCt valuesare used as quantitative measurement of the relative number of startingcopies of a particular target sequence in a nucleic acid sample. As oneCt unit corresponds to 1 PCR cycle or approximately a 2-fold relativeincrease relative to normal, two units corresponds to a 4-fold relativeincrease, 3 units corresponds to an 8-fold relative increase and so on,one can quantitatively measure the relative fold increase in mRNAexpression between two or more different tissues. The lower the Ct valuein a sample, the higher the starting copy number of that particulargene. If a standard curve is included in the assay, the relative amountof each target can be extrapolated and facilitates viewing of the dataas higher copy numbers also have relative quantities (as opposed tohigher copy numbers have lower Ct values) and also corrects for anyvariation of the generalized 1 Ct equals a 2 fold increase rule. Usingthis technique, the molecules listed below have been identified as beingsignificantly overexpressed (i.e., at least 2 fold) in a single (orlimited number) of specific tissue or cell types as compared to adifferent tissue or cell type (from both the same and different tissuedonors) with some also being identified as being significantlyoverexpressed (i.e., at least 2 fold) in cancerous cells when comparedto normal cells of the particular tissue or cell type, and thus,represent excellent polypeptide targets for therapy of cancer inmammals. Molecule specific expression in: as compared to: DNA225875(TAHO6) B cells non-B cells DNA225820 (TAHO25) B cells non-B cellsSummary

CD21/TAHO6 and CD19/TAHO25 expression levels in total RNA isolated frompurified B cells or from B cells from 20 B cell donors (310-1112)(AllCells) and averaged (Avg. B) was significantly higher thanrespective CD21/TAHO6 and CD19/TAHO25 expression levels in total RNAisolated from several white blood cell types, neutrophils (Neutr),natural killer cells (NK) (a T cell subset), dendritic cells (Dend),monocytes (Mono), CD4+T cells, CD8+T cells, CD34+ stem cells (data notshown).

Accordingly, as CD/21TAHO6 and CD19/TAHO25 are significantly expressedon B cells as compared to non-B cells as detected by TaqMan analysis,the molecules are excellent targets for therapy of tumors in mammals,including B-cell associated cancers, such as lymphomas (i.e.Non-Hodgkin's Lyphoma), leukemias (i.e. chronic lymphocytic leukemia),myelomas (i.e. multiple myeloma) and other cancers of hematopoieticcells.

Example 3 In Situ Hybridization

In situ hybridization is a powerful and versatile technique for thedetection and localization of nucleic acid sequences within cell ortissue preparations. It may be useful, for example, to identify sites ofgene expression, analyze the tissue distribution of transcription,identify and localize viral infection, follow changes in specific mRNAsynthesis and aid in chromosome mapping.

In situ hybridization is performed following an optimized version of theprotocol by Lu and Gillett, Cell Vision 1:169-176 (1994), usingPCR-generated ³³P-labeled riboprobes. Briefly, formalin-fixed,paraffin-embedded human tissues are sectioned, deparaffinized,deproteinated in proteinase K (20 g/ml) for 15 minutes at 37° C., andfurther processed for in situ hybridization as described by Lu andGillett, supra. A [³³-P] UTP-labeled antisense riboprobe is generatedfrom a PCR product and hybridized at 55° C. overnight. The slides aredipped in Kodak NTB2 nuclear track emulsion and exposed for 4 weeks.

³³P-Riboprobe Synthesis

6.0 μl (125 mCi) of ³³P-UTP (Amersham BF 1002, SA<2000 Ci/mmol) werespeed vac dried. To each tube containing dried ³³P-UTP, the followingingredients were added:

2.0 μl 5× transcription buffer

1.0 μl DTT (100 mM)

2.0 μl NTP mix (2.5 mM: 101; each of 10 mM GTP, CTP & ATP+10 μl H₂O)

1.0 μl UTP (50 μM)

1.0 μl Rnasin

1.0 μl DNA template (1 μg)

1.0 μl H₂O

1.0 μl RNA polymerase (for PCR products T3=AS, T7=S, usually)

The tubes are incubated at 37° C. for one hour. 1.0 μl RQ1 DNase areadded, followed by incubation at 37° C. for 15 minutes. 90 μl TE (10 mMTris pH 7.6/1 mM EDTA pH 8.0) are added, and the mixture is pipettedonto DE81 paper. The remaining solution is loaded in a Microcon-50ultrafiltration unit, and spun using program 10 (6 minutes). Thefiltration unit is inverted over a second tube and spun using program 2(3 minutes). After the final recovery spin, 100 μl TE is added. 1 μl ofthe final product is pipetted on DE81 paper and counted in 6 ml ofBiofluor II.

The probe is run on a TBE/urea gel. 1-3 μl of the probe or 5 μl of RNAMrk III are added to 3 μl of loading buffer. After heating on a 95° C.heat block for three minutes, the probe is immediately placed on ice.

The wells of gel are flushed, the sample loaded, and run at 180-250volts for 45 minutes. The gel is wrapped in saran wrap and exposed toXAR film with an intensifying screen in −70° C. freezer one hour toovernight.

³³P-Hybridization

A. Pretreatment of Frozen Sections

The slides are removed from the freezer, placed on aluminium trays andthawed at room temperature for 5 minutes. The trays are placed in 55° C.incubator for five minutes to reduce condensation. The slides are fixedfor 10 minutes in 4% paraformaldehyde on ice in the fume hood, andwashed in 0.5×SSC for 5 minutes, at room temperature (25 ml 20×SSC+975ml SQ H₂O). After deproteination in 0.5 μg/ml proteinase K for 10minutes at 37° C. (12.5 μl of 10 mg/ml stock in 250 ml prewarmedRNase-free RNAse buffer), the sections are washed in 0.5×SSC for 10minutes at room temperature. The sections are dehydrated in 70%, 95%,100% ethanol, 2 minutes each.

B. Pretreatment of Paraffin-Embedded Sections

The slides are deparaffinized, placed in SQ H₂O, and rinsed twice in2×SSC at room temperature, for 5 minutes each time. The sections aredeproteinated in 20 μg/ml proteinase K (500 μl of 10 mg/ml in 250 mlRNase-free RNase buffer; 37° C., 15 minutes)—human embryo, or 8×proteinase K (100 μl in 250 ml Rnase buffer, 37° C., 30minutes)—formalin tissues. Subsequent rinsing in 0.5×SSC and dehydrationare performed as described above.

C. Prehybridization

The slides are laid out in a plastic box lined with Box buffer (4×SSC,50% formamide)—saturated filter paper.

D. Hybridization

1.0×10⁶ cpm probe and 1.0 μl tRNA (50 mg/ml stock) per slide are heatedat 95° C. for 3 minutes. The slides are cooled on ice, and 48 μlhybridization buffer were added per slide. After vortexing, 50 μl ³³Pmix were added to 50 μl prehybridization on slide. The slides areincubated overnight at 55° C.

E. Washes

Washing is done 2×10 minutes with 2×SSC, EDTA at room temperature (400ml 20×SSC+16 ml 0.25M EDTA, V_(f)=4 L), followed by RNaseA treatment at37° C. for 30 minutes (500 μl of 10 mg/ml in 250 ml Rnase buffer=20μg/ml), The slides are washed 2×10 minutes with 2×SSC, EDTA at roomtemperature. The stringency wash conditions are as follows: 2 hours at55° C., 0.1×SSC, EDTA (20 ml 20×SSC+16 ml EDTA, V_(f)=4 L).

F. Oligonucleotides

In situ analysis is performed on a variety of DNA sequences disclosedherein. The oligonucleotides employed for these analyses are obtained soas to be complementary to the nucleic acids (or the complements thereof)as shown in the accompanying figures.

TAHO molecules that localize to particular B cells including lymphoidcells or to tissue sections from malignant lymphomas, includingHodgkin's lymphoma, follicular lymphoma, diffuse large cell lymphoma,small lymphocytic lymphoma and non-Hodgkin's lymphoma may be usefultargets for therapy of tumors in mammals, including B-cell associatedcancers, such as lymphomas (i.e. Non-Hodgkin's Lyphoma), leukemias (i.e.chronic lymphocytic leukemia), myelomas (i.e. multiple myeloma) andother cancers of hematopoietic cells.

Example 4 Use of TAHO as a Hybridization Probe

The following method describes use of a nucleotide sequence encodingTAHO as a hybridization probe for, i.e., detection of the presence ofTAHO in a mammal.

DNA comprising the coding sequence of full-length or mature TAHO asdisclosed herein can also be employed as a probe to screen forhomologous DNAs (such as those encoding naturally-occurring variants ofTAHO) in human tissue cDNA libraries or human tissue genomic libraries.

Hybridization and washing of filters containing either library DNAs isperformed under the following high stringency conditions. Hybridizationof radiolabeled TAHO-derived probe to the filters is performed in asolution of 50% formamide, 5×SSC, 0.1% SDS, 0.1% sodium pyrophosphate,50 mM sodium phosphate, pH 6.8, 2× Denhardt's solution, and 10% dextransulfate at 42° C. for 20 hours. Washing of the filters is performed inan aqueous solution of 0.1×SSC and 0.1% SDS at 42° C.

DNAs having a desired sequence identity with the DNA encodingfull-length native sequence TAHO can then be identified using standardtechniques known in the art.

Example 5 Expression of TAHO in E. coli

This example illustrates preparation of an unglycosylated form of TAHOby recombinant expression in E. coli.

The DNA sequence encoding TAHO is initially amplified using selected PCRprimers. The primers should contain restriction enzyme sites whichcorrespond to the restriction enzyme sites on the selected expressionvector. A variety of expression vectors may be employed. An example of asuitable vector is pBR322 (derived from E. coli; see Bolivar et al.,Gene, 2:95 (1977)) which contains genes for ampicillin and tetracyclineresistance. The vector is digested with restriction enzyme anddephosphorylated. The PCR amplified sequences are then ligated into thevector. The vector will preferably include sequences which encode for anantibiotic resistance gene, a trp promoter, a polyhis leader (includingthe first six STII codons, polyhis sequence, and enterokinase cleavagesite), the TAHO coding region, lambda transcriptional terminator, and anargU gene.

The ligation mixture is then used to transform a selected E. coli strainusing the methods described in Sambrook et al., supra. Transformants areidentified by their ability to grow on LB plates and antibioticresistant colonies are then selected. Plasmid DNA can be isolated andconfirmed by restriction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid culture medium such asLB broth supplemented with antibiotics. The overnight culture maysubsequently be used to inoculate a larger scale culture. The cells arethen grown to a desired optical density, during which the expressionpromoter is turned on.

After culturing the cells for several more hours, the cells can beharvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized TAHO protein can then be purified using a metalchelating column under conditions that allow tight binding of theprotein.

TAHO may be expressed in E. coli in a poly-His tagged form, using thefollowing procedure. The DNA encoding TAHO is initially amplified usingselected PCR primers. The primers will contain restriction enzyme siteswhich correspond to the restriction enzyme sites on the selectedexpression vector, and other useful sequences providing for efficientand reliable translation initiation, rapid purification on a metalchelation column, and proteolytic removal with enterokinase. ThePCR-amplified, poly-His tagged sequences are then ligated into anexpression vector, which is used to transform an E. coli host based onstrain 52 (W3110 fuhA(tonA) lon galE rpoHts(htpRts) clpP(lacIq).Transformants are first grown in LB containing 50 mg/ml carbenicillin at30° C. with shaking until an O.D.600 of 3-5 is reached. Cultures arethen diluted 50-100 fold into CRAP media (prepared by mixing 3.57 g(NH₄)₂SO₄, 0.71 g sodium citrate.2H2O, 1.07 g KCl, 5.36 g Difco yeastextract, 5.36 g Sheffield hycase SF in 500 mL water, as well as 110 mMMPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgSO₄) and grown forapproximately 20-30 hours at 30° C. with shaking. Samples are removed toverify expression by SDS-PAGE analysis, and the bulk culture iscentrifuged to pellet the cells. Cell pellets are frozen untilpurification and refolding.

E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) isresuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8buffer. Solid sodium sulfite and sodium tetrathionate is added to makefinal concentrations of 0.1M and 0.02 M, respectively, and the solutionis stirred overnight at 4° C. This step results in a denatured proteinwith all cysteine residues blocked by sulfitolization. The solution iscentrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. Thesupernatant is diluted with 3-5 volumes of metal chelate column buffer(6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micronfilters to clarify. The clarified extract is loaded onto a 5 ml QiagenNi-NTA metal chelate column equilibrated in the metal chelate columnbuffer. The column is washed with additional buffer containing 50 mMimidazole (Calbiochem, Utrol grade), pH 7.4. The protein is eluted withbuffer containing 250 mM imidazole. Fractions containing the desiredprotein are pooled and stored at 4° C. Protein concentration isestimated by its absorbance at 280 nm using the calculated extinctioncoefficient based on its amino acid sequence.

The proteins are refolded by diluting the sample slowly into freshlyprepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCl,2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. Refoldingvolumes are chosen so that the final protein concentration is between 50to 100 micrograms/ml. The refolding solution is stirred gently at 4° C.for 12-36 hours. The refolding reaction is quenched by the addition ofTFA to a final concentration of 0.4% (pH of approximately 3). Beforefurther purification of the protein, the solution is filtered through a0.22 micron filter and acetonitrile is added to 2-10% finalconcentration. The refolded protein is chromatographed on a Poros R1/Hreversed phase column using a mobile buffer of 0.1% TFA with elutionwith a gradient of acetonitrile from 10 to 80%. Aliquots of fractionswith A280 absorbance are analyzed on SDS polyacrylamide gels andfractions containing homogeneous refolded protein are pooled. Generally,the properly refolded species of most proteins are eluted at the lowestconcentrations of acetonitrile since those species are the most compactwith their hydrophobic interiors shielded from interaction with thereversed phase resin. Aggregated species are usually eluted at higheracetonitrile concentrations. In addition to resolving misfolded forms ofproteins from the desired form, the reversed phase step also removesendotoxin from the samples.

Fractions containing the desired folded TAHO polypeptide are pooled andthe acetonitrile removed using a gentle stream of nitrogen directed atthe solution. Proteins are formulated into 20 mM Hepes, pH 6.8 with 0.14M sodium chloride and 4% mannitol by dialysis or by gel filtration usingG25 Superfine (Pharmacia) resins equilibrated in the formulation bufferand sterile filtered.

Certain of the TAHO polypeptides disclosed herein have been successfullyexpressed and purified using this technique(s).

Example 6 Expression of TAHO in Mammalian Cells

This example illustrates preparation of a potentially glycosylated formof TAHO by recombinant expression in mammalian cells.

The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employedas the expression vector. Optionally, the TAHO DNA is ligated into pRK5with selected restriction enzymes to allow insertion of the TAHO DNAusing ligation methods such as described in Sambrook et al., supra. Theresulting vector is called pRK5-TAHO.

In one embodiment, the selected host cells may be 293 cells. Human 293cells (ATCC CCL 1573) are grown to confluence in tissue culture platesin medium such as DMEM supplemented with fetal calf serum andoptionally, nutrient components and/or antibiotics. About 10 μgpRK5-TAHO DNA is mixed with about 1 μg DNA encoding the VA RNA gene[Thimmappaya et al., Cell, 31:543 (1982)] and dissolved in 500 μl of 1nM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl₂. To this mixture is added,dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO₄,and a precipitate is allowed to form for 10 minutes at 25° C. Theprecipitate is suspended and added to the 293 cells and allowed tosettle for about four hours at 37° C. The culture medium is aspiratedoff and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293cells are then washed with serum free medium, fresh medium is added andthe cells are incubated for about 5 days.

Approximately 24 hours after the transfections, the culture medium isremoved and replaced with culture medium (alone) or culture mediumcontaining 200 μCi/ml ³⁵S-cysteine and 200 μCi/ml ³⁵S-methionine. Aftera 12 hour incubation, the conditioned medium is collected, concentratedon a spin filter, and loaded onto a 15% SDS gel. The processed gel maybe dried and exposed to film for a selected period of time to reveal thepresence of TAHO polypeptide. The cultures containing transfected cellsmay undergo further incubation (in serum free medium) and the medium istested in selected bioassays.

In an alternative technique, TAHO may be introduced into 293 cellstransiently using the dextran sulfate method described by Somparyrac etal., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells are grown tomaximal density in a spinner flask and 700 μg pRK5-TAHO DNA is added.The cells are first concentrated from the spinner flask bycentrifugation and washed with PBS. The DNA-dextran precipitate isincubated on the cell pellet for four hours. The cells are treated with20% glycerol for 90 seconds, washed with tissue culture medium, andre-introduced into the spinner flask containing tissue culture medium, 5μg/ml bovine insulin and 0.1 μg/ml bovine transferrin. After about fourdays, the conditioned media is centrifuged and filtered to remove cellsand debris. The sample containing expressed TAHO can then beconcentrated and purified by any selected method, such as dialysisand/or column chromatography.

In another embodiment, TAHO can be expressed in CHO cells. The pRK5-TAHOcan be transfected into CHO cells using known reagents such as CaPO₄ orDEAE-dextran. As described above, the cell cultures can be incubated,and the medium replaced with culture medium (alone) or medium containinga radiolabel such as ³⁵S-methionine. After determining the presence ofTAHO polypeptide, the culture medium may be replaced with serum freemedium. Preferably, the cultures are incubated for about 6 days, andthen the conditioned medium is harvested. The medium containing theexpressed TAHO can then be concentrated and purified by any selectedmethod.

Epitope-tagged TAHO may also be expressed in host CHO cells. The TAHOmay be subcloned out of the pRK5 vector. The subclone insert can undergoPCR to fuse in frame with a selected epitope tag such as a poly-his taginto a Baculovirus expression vector. The poly-his tagged TAHO insertcan then be subcloned into a SV40 driven vector containing a selectionmarker such as DHFR for selection of stable clones. Finally, the CHOcells can be transfected (as described above) with the SV40 drivenvector. Labeling may be performed, as described above, to verifyexpression. The culture medium containing the expressed poly-His taggedTAHO can then be concentrated and purified by any selected method, suchas by Ni²⁺-chelate affinity chromatography.

TAHO may also be expressed in CHO and/or COS cells by a transientexpression procedure or in CHO cells by another stable expressionprocedure.

Stable expression in CHO cells is performed using the followingprocedure. The proteins are expressed as an IgG construct(immunoadhesin), in which the coding sequences for the soluble forms(e.g. extracellular domains) of the respective proteins are fused to anIgG1 constant region sequence containing the hinge, CH2 and CH2 domainsand/or is a poly-His tagged form.

Following PCR amplification, the respective DNAs are subcloned in a CHOexpression vector using standard techniques as described in Ausubel etal., Current Protocols of Molecular Biology, Unit 3.16, John Wiley andSons (1997). CHO expression vectors are constructed to have compatiblerestriction sites 5′ and 3′ of the DNA of interest to allow theconvenient shuttling of cDNA's. The vector used expression in CHO cellsis as described in Lucas et al., Nucl. Acids Res. 24:9 (1774-1779(1996), and uses the SV40 early promoter/enhancer to drive expression ofthe cDNA of interest and dihydrofolate reductase (DHFR). DHFR expressionpermits selection for stable maintenance of the plasmid followingtransfection.

Twelve micrograms of the desired plasmid DNA is introduced intoapproximately 10 million CHO cells using commercially availabletransfection reagents Superfect® (Quiagen), Dosper® or Fugene®(Boehringer Mannheim). The cells are grown as described in Lucas et al.,supra. Approximately 3×10⁷ cells are frozen in an ampule for furthergrowth and production as described below.

The ampules containing the plasmid DNA are thawed by placement intowater bath and mixed by vortexing. The contents are pipetted into acentrifuge tube containing 10 mLs of media and centrifuged at 1000 rpmfor 5 minutes. The supernatant is aspirated and the cells areresuspended in 10 mL of selective media (0.2 μm filtered PS20 with 5%0.2 μm diafiltered fetal bovine serum). The cells are then aliquotedinto a 100 mL spinner containing 90 mL of selective media. After 1-2days, the cells are transferred into a 250 mL spinner filled with 150 mLselective growth medium and incubated at 37° C. After another 2-3 days,250 mL, 500 mL and 2000 mL spinners are seeded with 3×10⁵ cells/mL. Thecell media is exchanged with fresh media by centrifugation andresuspension in production medium. Although any suitable CHO media maybe employed, a production medium described in U.S. Pat. No. 5,122,469,issued Jun. 16, 1992 may actually be used. A 3 L production spinner isseeded at 1.2×10⁶ cells/mL. On day 0, the cell number pH is determined.On day 1, the spinner is sampled and sparging with filtered air iscommenced. On day 2, the spinner is sampled, the temperature shifted to33° C., and 30 mL of 500 g/L glucose and 0.6 mL of 10% antifoam (e.g.,35% polydimethylsiloxane emulsion, Dow Corning 365 Medical GradeEmulsion) taken. Throughout the production, the pH is adjusted asnecessary to keep it at around 7.2. After 10 days, or until theviability dropped below 70%, the cell culture is harvested bycentrifugation and filtering through a 0.22 μm filter. The filtrate waseither stored at 4° C. or immediately loaded onto columns forpurification.

For the poly-His tagged constructs, the proteins are purified using aNi-NTA column (Qiagen).

Before purification, imidazole is added to the conditioned media to aconcentration of 5 mM. The conditioned media is pumped onto a 6 mlNi-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4° C. Afterloading, the column is washed with additional equilibration buffer andthe protein eluted with equilibration buffer containing 0.25 Mimidazole.

The highly purified protein is subsequently desalted into a storagebuffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, witha 25 ml G25 Superfine (Pharmacia) column and stored at −80° C.

Immunoadhesin (Fc-containing) constructs are purified from theconditioned media as follows. The conditioned medium is pumped onto a 5ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Naphosphate buffer, pH 6.8. After loading, the column is washedextensively with equilibration buffer before elution with 100 mM citricacid, pH 3.5. The eluted protein is immediately neutralized bycollecting 1 ml fractions into tubes containing 275 μL of 1 M Trisbuffer, pH 9. The highly purified protein is subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity is assessed by SDS polyacrylamide gels and by N-terminalamino acid sequencing by Edman degradation.

Certain of the TAHO polypeptides disclosed herein have been successfullyexpressed and purified using this technique(s).

Example 7 Expression of TAHO in Yeast

The following method describes recombinant expression of TAHO in yeast.

First, yeast expression vectors are constructed for intracellularproduction or secretion of TAHO from the ADH2/GAPDH promoter. DNAencoding TAHO and the promoter is inserted into suitable restrictionenzyme sites in the selected plasmid to direct intracellular expressionof TAHO. For secretion, DNA encoding TAHO can be cloned into theselected plasmid, together with DNA encoding the ADH2/GAPDH promoter, anative TAHO signal peptide or other mammalian signal peptide, or, forexample, a yeast alpha-factor or invertase secretory signal/leadersequence, and linker sequences (if needed) for expression of TAHO.

Yeast cells, such as yeast strain AB110, can then be transformed withthe expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

Recombinant TAHO can subsequently be isolated and purified by removingthe yeast cells from the fermentation medium by centrifugation and thenconcentrating the medium using selected cartridge filters.

The concentrate containing TAHO may further be purified using selectedcolumn chromatography resins. Certain of the TAHO polypeptides disclosedherein have been successfully expressed and purified using thistechnique(s).

Example 8 Expression of TAHO in Baculovirus-Infected Insect Cells

The following method describes recombinant expression of TAHO inBaculovirus-infected insect cells.

The sequence coding for TAHO is fused upstream of an epitope tagcontained within a baculovirus expression vector. Such epitope tagsinclude poly-his tags and immunoglobulin tags (like Fc regions of IgG).A variety of plasmids may be employed, including plasmids derived fromcommercially available plasmids such as pVL1393 (Novagen). Briefly, thesequence encoding TAHO or the desired portion of the coding sequence ofTAHO such as the sequence encoding an extracellular domain of atransmembrane protein or the sequence encoding the mature protein if theprotein is extracellular is amplified by PCR with primers complementaryto the 5′ and 3′ regions. The 5′ primer may incorporate flanking(selected) restriction enzyme sites. The product is then digested withthose selected restriction enzymes and subcloned into the expressionvector.

Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4-5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression are performed as described byO'Reilley et al., Baculovirus expression vectors: A Laboratory Manual,Oxford: Oxford University Press (1994).

Expressed poly-his tagged TAHO can then be purified, for example, byNi²⁺-chelate affinity chromatography as follows. Extracts are preparedfrom recombinant virus-infected Sf9 cells as described by Rupert et al.,Nature, 362:175-179 (1993). Briefly, Sf9 cells are washed, resuspendedin sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl₂; 0.1 mM EDTA;10% glycerol; 0.1% NP-40; 0.4 M KCl), and sonicated twice for 20 secondson ice. The sonicates are cleared by centrifugation, and the supernatantis diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10%glycerol, pH 7.8) and filtered through a 0.45 μm filter. A Ni²⁺-NTAagarose column (commercially available from Qiagen) is prepared with abed volume of 5 mL, washed with 25 mL of water and equilibrated with 25mL of loading buffer. The filtered cell extract is loaded onto thecolumn at 0.5 mL per minute. The column is washed to baseline A₂₈₀ withloading buffer, at which point fraction collection is started. Next, thecolumn is washed with a secondary wash buffer (50 mM phosphate; 300 mMNaCl, 10% glycerol, pH 6.0), which elutes nonspecifically bound protein.After reaching A₂₈₀ baseline again, the column is developed with a 0 to500 mM Imidazole gradient in the secondary wash buffer. One mL fractionsare collected and analyzed by SDS-PAGE and silver staining or Westernblot with Ni²⁺-NTA-conjugated to alkaline phosphatase (Qiagen).Fractions containing the eluted His₁₀-tagged TAHO are pooled anddialyzed against loading buffer.

Alternatively, purification of the IgG tagged (or Fc tagged) TAHO can beperformed using known chromatography techniques, including for instance,Protein A or protein G column chromatography.

Certain of the TAHO polypeptides disclosed herein have been successfullyexpressed and purified using this technique(s).

Example 9 Preparation of Antibodies that Bind TAHO

This example illustrates preparation of monoclonal antibodies which canspecifically bind TAHO.

Techniques for producing the monoclonal antibodies are known in the artand are described, for instance, in Goding, supra. Immunogens that maybe employed include purified TAHO, fusion proteins containing TAHO, andcells expressing recombinant TAHO on the cell surface. Selection of theimmunogen can be made by the skilled artisan without undueexperimentation.

Mice, such as Balb/c, are immunized with the TAHO immunogen emulsifiedin complete Freund's adjuvant and injected subcutaneously orintraperitoneally in an amount from 1-100 micrograms. Alternatively, theimmunogen is emulsified in MPL-TDM adjuvant (Ribi ImmunochemicalResearch, Hamilton, Mont.) and injected into the animal's hind footpads. The immunized mice are then boosted 10 to 12 days later withadditional immunogen emulsified in the selected adjuvant. Thereafter,for several weeks, the mice may also be boosted with additionalimmunization injections. Serum samples may be periodically obtained fromthe mice by retro-orbital bleeding for testing in ELISA assays to detectanti-TAHO antibodies.

After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of immunogen. Three to four days later, the mice aresacrificed and the spleen cells are harvested. The spleen cells are thenfused (using 35% polyethylene glycol) to a selected murine myeloma cellline such as P3X63AgU.1, available from ATCC, No. CRL 1597. The fusionsgenerate hybridoma cells which can then be plated in 96 well tissueculture plates containing HAT (hypoxanthine, aminopterin, and thymidine)medium to inhibit proliferation of non-fused cells, myeloma hybrids, andspleen cell hybrids.

The hybridoma cells will be screened in an ELISA for reactivity againstimmunogen. Determination of “positive” hybridoma cells secreting thedesired monoclonal antibodies against immunogen is within the skill inthe art.

The positive hybridoma cells can be injected intraperitoneally intosyngeneic Balb/c mice to produce ascites containing the anti-immunogenmonoclonal antibodies. Alternatively, the hybridoma cells can be grownin tissue culture flasks or roller bottles. Purification of themonoclonal antibodies produced in the ascites can be accomplished usingammonium sulfate precipitation, followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can be employed.

Antibodies directed against certain of the TAHO polypeptides disclosedherein can be successfully produced using this technique(s). Morespecifically, functional monoclonal antibodies that are capable ofrecognizing and binding to TAHO protein (as measured by standard ELISA,FACS sorting analysis and/or immunohistochemistry analysis) can besuccessfully generated against the following TAHO proteins as disclosedherein: TAHO6 (DNA225875), TAHO 25 (DNA225820) and TAHO41 (DNA548572).

In addition to the preparation of monoclonal antibodies directed againstthe TAHO polypeptides as described herein, many of the monoclonalantibodies can be successfully conjugated to a cell toxin for use indirecting the cellular toxin to a cell (or tissue) that expresses a TAHOpolypeptide of interested (both in vitro and in vivo). For example,toxin (e.g., DM1) derivitized monoclonal antibodies can be successfullygenerated to the following TAHO polypeptides as described herein: TAHO6(DNA225875), TAHO 25 (DNA225820) and TAHO41 (DNA548572).

Example 10 Purification of TAHO Polypeptides Using Specific Antibodies

Native or recombinant TAHO polypeptides may be purified by a variety ofstandard techniques in the art of protein purification. For example,pro-TAHO polypeptide, mature TAHO polypeptide, or pre-TAHO polypeptideis purified by immunoaffinity chromatography using antibodies specificfor the TAHO polypeptide of interest. In general, an immunoaffinitycolumn is constructed by covalently coupling the anti-TAHO polypeptideantibody to an activated chromatographic resin.

Polyclonal immunoglobulins are prepared from immune sera either byprecipitation with ammonium sulfate or by purification on immobilizedProtein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise,monoclonal antibodies are prepared from mouse ascites fluid by ammoniumsulfate precipitation or chromatography on immobilized Protein A.Partially purified immunoglobulin is covalently attached to achromatographic resin such as CnBr-activated SEPHAROSE™ (Pharmacia LKBBiotechnology). The antibody is coupled to the resin, the resin isblocked, and the derivative resin is washed according to themanufacturer's instructions.

Such an immunoaffinity column is utilized in the purification of TAHOpolypeptide by preparing a fraction from cells containing TAHOpolypeptide in a soluble form. This preparation is derived bysolubilization of the whole cell or of a subcellular fraction obtainedvia differential centrifugation by the addition of detergent or by othermethods well known in the art. Alternatively, soluble TAHO polypeptidecontaining a signal sequence may be secreted in useful quantity into themedium in which the cells are grown.

A soluble TAHO polypeptide-containing preparation is passed over theimmunoaffinity column, and the column is washed under conditions thatallow the preferential absorbance of TAHO polypeptide (e.g., high ionicstrength buffers in the presence of detergent). Then, the column iseluted under conditions that disrupt antibody/TAHO polypeptide binding(e.g., a low pH buffer such as approximately pH 2-3, or a highconcentration of a chaotrope such as urea or thiocyanate ion), and TAHOpolypeptide is collected.

Example 11 In Vitro Tumor Cell Killing Assay

A. In Vitro Tumor Cell Killing in Non-Transfected Cells

Mammalian cells expressing the TAHO polypeptide of interest may beobtained using standard expression vector and cloning techniques.Alternatively, many tumor cell lines expressing TAHO polypeptides ofinterest are publicly available, for example, through the ATCC and canbe routinely identified using standard ELISA or FACS analysis. Anti-TAHOpolypeptide monoclonal antibodies (commercially available and toxinconjugated derivatives thereof) may then be employed in assays todetermine the ability of the antibody to kill TAHO polypeptideexpressing cells in vitro.

For example, cells expressing the TAHO polypeptide of interest areobtained as described above and plated into 96 well dishes. In oneanalysis, the antibody/toxin conjugate (or naked antibody) is includedthroughout the cell incubation for a period of 4 days. In a secondindependent analysis, the cells are incubated for 1 hour with theantibody/toxin conjugate (or naked antibody) and then washed andincubated in the absence of antibody/toxin conjugate for a period of 4days. Cell viability is then measured using the CellTiter-GloLuminescent Cell Viability Assay from Promega (Cat#G7571). Untreatedcells serve as a negative control.

B cell lines (ARH-77, BJAB, Daudi, DOHH-2, Su-DHL-4, Raji and Ramos)were prepared at 5000 cells/well in separate sterile round bottom 96well tissue culture treated plates (Cellstar 650 185). Cells were inassay media (RPMI 1460, 1% L-Glutamine (Promega Glutamax), 10% fetalbovine serum (FBS; from Hyclone) and 10 mM HEPES). Cells wereimmediately placed in a 37° C. incubator overnight. Antibody drugconjugates (using commercially available anti-CD19, anti-CD20,anti-CD21, anti-CD79A, anti-CD79B) were diluted at 2×10 μg/ml in assaymedium. Conjugates were linked with crosslinkers SMCC or disulfidelinker SPP to DM1 toxin. Further, conjugates may be linked with Vc-PABto MMAE toxin. Herceptin based conjugates (SMCC-DM1 or SPP-DM1) wereused as negative controls. Free L-DM1 equivalent to the conjugateloading dose was used as a positive control. Samples were vortexed toensure homogenous mixture prior to dilution. The antibody drugconjugates were further diluted serially 1:3. The cell lines were loaded50 μl of each sample per row using a Rapidplate® 96/384 Zymarkautomation system. When the entire plate was loaded, the plates werereincubated for 3 days to permit the toxins to take effect. Thereactions were assessed for cell viability using the CellTiterGlo kit(Promega) where the reactions were stopped by applying 100 μl/well ofCell Glo (Promega, Cat. #G7571/2/3) to all the wells for 10 minutes. The100 μl of the stopped well were transferred into 96 well white tissueculture treated plates, clear bottom (Costar 3610) and the luminescencewas read and reported as relative light units (RLU). TAHO antibodies forthis experiment included commercially available antibodies, includinganti-TAHO6/CD21 (ATCC HB5) and anti-TAHO25/CD19 (Biomeda CB-19).

Summary

(1) Anti-TAHO25/CD19 antibody conjugated to DM1 toxin (CD19-SPP-DM1 andCD19-SMCC-DM1) showed significant tumor cell killing when compared toanti-TAHO25/CD19 antibody alone or negative control anti-HER2 conjugatedto DM1 toxin (anti-HER2-SMCC-DM1) in RAJI or RAMOS cells. Further,greater tumor cell killing was observed with CD19-SMCC-DM1 compared toCD19-SPP-DM1.

(2) Anti-TAHO6/CD21 antibody conjugated to DM1 toxin (CD21-SPP-DM1 andCD21-SMCC-DM1) showed weak tumor cell killing when compared toanti-TAHO6/CD21 antibody alone or negative control anti-HER2 conjugatedto DM1 toxin (anti-HER2-SMCC-DM1) in RAJI or RAMOS cells. Further,greater tumor cell killing was observed with CD21-SPP-DM1 compared toCD21-SMCC-DM1.

B. In Vitro Tumor Cell Killing in Cells Transfected with TAHO41/CD21Variant

For further analysis, a Ramos B-cell line expressing a one amino-acidvariant of TAHO6 (DNA225875; CD21), herein referred to as TAHO41(DNA548572; CD21 variant), was used in a three-day cytotoxicity assay.

For expression of TAHO41, full length CD21 (Swiss-Prot P20023) wascloned into pCMV.PD5.IREs-eGFP vector at XbaI and ApaI sites. Forgeneration of the expression vector, the extracellular domain of CD21with a C-terminal FLAG tag was excised from a pRK vector containing theextracellular domain of CD21 with a C-terminal FLAG tag and subclonedinto pIRES2-dsRed2 (Clontech), using NcoI and XhoI, both blunted,generating CD21 (EDC)-FLAG/pIRES2-dsRed2. The transmembrane domain andcytoplasmic tail of CD21 was obtained by RT-PCR from RAJI cell RNA usingBamHI flanked primers and cloned into CD21 (ECD)-FLAG/pIRES2-dsRed2,generating untagged full-length CD21 (FL)/pIRES2-dsRed2. The full-lengthCD21 was then excised from CD21 (FL)/pIRES2-dsRed2 and cloned intopCMV.PD5.IREs-eGFP cut with XbaI and ApaI, generating CD21(FL)/pCMV-PD5.IREs-eGFP. For transfection of TAHO41 (CD21 variant;DNA548572), 2×10⁶ Ramos cells (Passage 6) were spun down and resuspendedin 100 μl Solution T from Amaxa Nucleofector Kit, followed by 2 μg CD21(CD21 (FL)/pCMV.PD5.IREs-eGFP vector) DNA and put in the plastic cuvetteprovided with Amaxa kit. Cells were then electroporated using program0-06 in a Amaxa Nucleofector II. Cells were then placed in 1 well of12-well dish with 1.5 ml B-cell growth medium (RPMI with 10% HycloneFBS, 1% L-glutamine), along with 0.5 ml growth medium used to rinse thecuvette, and incubated at 37° C. in the tissue culture incubator. After48 hours, the media was replaced with 2.0 ml B-cell growth mediumcontaining 0.5 μg/ml puromycin and 1 mM Na pyruvate. Positive clonesbegan to grow after 18-24 days of puromycin selection. TwoCD21-expressing pools of cells (herein referred to as CD21 “Ramos clone1” and CD21 “Ramos clone 3”) were obtained out of a total 10 wells. HighCD21 expressors from each pool were then further selected by FACSsorting using Alexa488-labeled anti-CD21 (ATCC HB-135) antibodies.

The cells from CD21 Ramos clone 1 expressed CD21 at a mean fluorescentintensity (MFI) of about 160 and CD19 at a MFI of about 40, the cellsfrom CD21 Ramos clone 3 expressed CD21 less at a MFI of about 40 andCD19 at a MFI of about 60, and the untransfected Ramos cells did notexpress CD21 (MFI of about 0) and expressed CD19 at a MFI of about 80.Untransfected Raji cells expressed CD21 at an MFI of about 100 and CD19at an MFI of about 200 (FIG. 9).

B cell line (Ramos) and B cell lines expressing CD21 (CD21 “Ramos clone1” were prepared as described above and tested in the cytotoxicity assayas described above in Example 11A.

Summary

(1) Anti-TAHO25/CD19 antibody conjugated to DM1 toxin (CD19-SMCC-DM1)showed greater tumor cell killing in untransfected Ramos cells (notexpressing CD21) when compared to tumor cell killing in CD21 “Ramosclone 1” (expressing CD21) cells. Specifically, after treatment with 3.3μg/ml anti-TAHO25/CD19 antibody conjugated to DM1 toxin (CD19-SMCC-DM1),95% of the CD21 “Ramos clone 1” cells were alive, while 20% ofuntransfected Ramos cells were alive after the three day incubation.

Anti-TAHO polypeptide monoclonal antibodies are useful for reducing invitro tumor growth of tumors, including B-cell associated cancers, suchas lymphomas (i.e. Non-Hodgkin's Lyphoma), leukemias (i.e. chroniclymphocytic leukemia), myelomas (i.e. multiple myeloma) and othercancers of hematopoietic cells. Further, anti-TAHO25/CD19 antibodyconjugated to DM1 toxin (i.e. via an SMCC linker) may be more effectivein reducing in vitro tumor growth of tumors, including B-cell associatedcancers, such as lymphomas (i.e. Non-Hodgkin's Lyphoma), leukemias (i.e.chronic lymphocytic leukemia), myelomas (i.e. multiple myeloma) andother cancers of hematopoietic cells, that are expressing low amounts ofCD21 or are CD21 negative.

Example 12 In Vivo Tumor Cell Killing Assay

To test the efficacy of conjugated or unconjugated anti-TAHO polypeptidemonoclonal antibodies, the effect of anti-TAHO antibody on tumors inmice were analyzed. Female CB17 ICR SCID mice (6-8 weeks of age fromCharles Rivers Laboratories; Hollister, Calif.) were inoculatedsubcutaneously with 5×10⁶ RAJI cells or 2×10⁷ BJAB-luciferase cells.Tumor volume was calculated based on two dimensions, measured usingcalipers, and was expressed in mm³ according to the formula: V=0.5a×b²,where a and b are the long and the short diameters of the tumor,respectively. Data collected from each experimental group were expressedas mean ±SE. Mice were separated into groups of 8-10 mice with a meantumor volume between 100-200 mm³, at which point intravenous (i.v.)treatment began at the antibody dose of 5 mg/kg weekly for two to threeweeks. Tumors were measured either once or twice a week throughout theexperiment. Mice were euthanized before tumor volumes reached 3000 mm³or when tumors showed signs of impending ulceration.

All animal protocols were approved by an Institutional Animal Care andUse Committee (IACUC). Linkers between the antibody and the toxin thatwere used were SPP, SMCC or cys-MC-vc-PAB (a valine-citrulline (vc)dipeptide linker reagent having a maleimide component and apara-aminobenzylcarbamoyl (PAB) self-immolative component. Toxins usedwere DM1 or MMAE. TAHO antibodies for this experiment includedcommercially available antibodies, including anti-TAHO6/CD21 (ATCC HB135) and anti-TAHO25/CD19 (Biomeda CB-19).

Summary

(1) Anti-TAHO6/CD21 antibody conjugated with DM1 toxin(anti-CD21-SPP-DM1) showed inhibition of tumor growth in SCID mice withRAJI tumors when treated weekly with 5 mg/kg of antibody compared toanti-CD21 antibodies and herceptin antibodies conjugated to DM1 toxin(anti-Herceptin-SMCC-DM1 and anti-Herceptin-SPP-DM1). Specifically, atday 19, 8 out of 8 mice treated with anti-CD21-SPP-DM1 showed completeregression of tumors. At day 19, 8 out of 8 mice treated with anti-CD21,anti-herceptin-SPP-DM1, anti-herceptin-SMCC-DM1 or anti-CD21-SMCC-DM1showed tumor incidence. At day 19, 7 out of 8 mice treated withanti-CD20-SMCC-DM1 antibody showed tumor incidence.

(2) Anti-TAHO6/CD21 antibody conjugated with MMAE toxin(anti-CD21-cys-Mc-vc-PAB-MMAE) showed inhibition of tumor growth in SCIDmice with RAJI tumors when treated with 5 mg/kg of antibody compared tonegative control anti-CD1 antibody or anti-herceptin antibody.Specifically at day 14, 5 out of 9 mice treated withanti-CD21-cys-MC-vc-PAB-MMAE showed partical regression of tumors and 4out of 9 mice treated with anti-CD21-cys-MC-vc-PAB-MMAE showed completeregression of tumors. At day 14, 10 out of 10 mice treated withanti-herceptin or anti-CD21 antibody showed tumor incidence.

(3) Anti-TAHO25/CD19 antibody conjugated with DM1 toxin(anti-CD19-SPP-DM1) showed inhibition of tumor growth in SCID mice withRAJI tumors when treated with 5 mg/kg of antibody compared to negativecontrol anti-CD19 antibody conjugated to DM1 (anti-CD19-SMCC-DM1),anti-CD22 antibody conjugated to DM1 (anti-CD22-SMCC-DM1) andanti-herceptin antibody conjugated to DM1 (anti-herceptin-smcc-DM1 oranti-herceptin-spp-DM1). Specifically at day 14, 2 out of 6 mice treatedwith anti-CD19-SPP-DM1 showed partical regression of tumors and 3 out of6 mice treated with anti-CD19-SPP-DM1 showed complete regression oftumors. At day 14, 8 out of 8 mice treated with anti-herceptin-SPP-DM1,anti-herceptin-SMCC-DM1, anti-CD19-SMCC-DM1 or anti-CD22-SMCC-DM1 showedtumor incidence.

Anti-TAHO polypeptide monoclonal antibodies are useful for reducing invivo tumor growth of tumors in mammals, including B-cell associatedcancers, such as lymphomas (i.e. Non-Hodgkin's Lyphoma), leukemias (i.e.chronic lymphocytic leukemia), myelomas (i.e. multiple myeloma) andother cancers of hematopoietic cells.

Example 13 Immunohistochemistry

To determine tissue expression of TAHO polypeptide and to confirm themicroarray results from Example 1, immunohistochemical detection of TAHOpolypeptide expression was examined in snap-frozen and formalin-fixedparaffin-embedded (FFPE) lymphoid tissues, including palatine tonsil,spleen, lymph node and Peyer's patches from the Genentech Human TissueBank.

Prevalence of TAHO target expression was evaluated on FFPE lymphomatissue microarrays (Cybrdi) and a panel of 24 frozen human lymphomaspecimens. Frozen tissue specimens were sectioned at 5 μm, air-dried andfixed in acetone for 5 minutes prior to immunostaining.Paraffin-embedded tissues were sectioned at 5 μm and mounted onSuperFrost Plus microscope slides (VWR).

For frozen sections, slides were placed in TBST, 1% BSA and 10% normalhorse serum containing 0.05% sodium azide for 30 minutes, then incubatedwith Avidin/Biotin blocking kit (Vector) reagents before addition ofprimary antibody. Mouse monoclonal primary antibodies (commerciallyavailable) were detected with biotinylated horse anti-mouse IgG(Vector), followed by incubation in Avidin-Biotin peroxidase complex(ABC Elite, Vector) and metal-enhanced diaminobenzidinetetrahydrochloride (DAB, Pierce). Control sections were incubated withisotype-matched irrelevant mouse monoclonal antibody (Pharmingen) atequivalent concentration. Following application of the ABC-HRP reagent,sections were incubated with biotinyl-tyramide (Perkin Elmer) inamplification diluent for 5-10 minutes, washed, and again incubated withC ABC-HRP reagent. Detection was using DAB as described above.

FFPE human tissue sections were dewaxed into distilled water, treatedwith Target Retrieval solution (Dako) in a boiling water bath for 20minutes, followed by a 20 minute cooling period. Residual endogenousperoxidase activity was blocked using 1× Blocking Solution (KPL) for 4minutes. Sections were incubated with Avidin/Biotin blocking reagentsand Blocking Buffer containing 10% normal horse serum before addition ofthe monoclonal antibodies, diluted to 0.5-5.0 μg/ml in Blocking Buffer.Sections were then incubated sequentially with biotinylated anti-mousesecondary antibody, followed by ABC-HRP and chromogenic detection withDAB. Tyramide Signal Amplification, described above, was used toincrease sensitivity of staining for a number of TAHO targets (CD21).

For dual immunofluorescent staining, frozen sections were incubated witha mixture of anti-CD19 antibody (clone SJ25-C1; Southern Biotechnology;mouse-IgG1 isotype) and anti-CD21 antibody (clone HB135; ATC; mouseIgG2a isotype), followed by a mixture of FITC-conjugated goat anti-mouseIgG1 and TRITC-conjugated goat anti-mouse IgG2a (SouthernBiotechnology). Nuclei were counterstained with DAPI and sections werecoverslipped with ProLong Gold antifade (Molecular Probes). Slides wereviewed and imaged on an Olympus BX51 microscope equipped withDAPI/FITC/TRITC filter sets and a Hamamatsu CCD camera. Images wereacquired using MetaMorph software (Molecular Devices).

Summary

(1) TAHO6 (CD21) showed strong labeling of follicular dendritic cells ingerminal centers and mature B cells within mantle zone as detected withclone HB-135 (ATCC) in FFPE human tonsil tissue and using tyramidesignal amplification (TSA) (data not shown).

(2) TAHO6 (CD21) was not detected in 25% of CD20+FFPE samples of diffuselarge B-cell lymphomas (DLBCL) and 29% of B-cell lymphomas. CD21 wasweakly detected (1+) in 33% of CD20+FFPE samples of diffuse large B-celllymphomas (DLBCL) and 29% of B-cell lymphoma.

(3) TAHO6 (CD21) detection was less than CD19 detection (CD19>CD21) in38% of CD19+ frozen samples of diffuse large B-cell lymphomas (DLBCL;n=8). Further, 25% of CD19+ frozen samples of diffuse large B-celllymphomas (DLBCL; n=8) were CD21−. Additionally, TAHO6 (CD21) detectionwas less than CD19 detection (CD19>CD21) in 29% of CD19+ frozen samplesof low grade Non-Hodgkin's Lymphoma (NHL; n=7).

Even further, several of the CD21+ samples had CD21 expression mainlyrestricted to follicles, and absent from the majority of CD19-expressingB-cells in the samples. Thus, some of the samples classified asCD21<CD19 may be eligible for treatment with anti-CD19 ADCs whichrequire internalization for efficacy (i.e. anti-CD19-SMCC-DM1) if theCD21 expression is restricted and absent from a majority ofCD19-expressing B cells in a tumor sample (as detectable viaimmunohistochemistry).

Accordingly, in light of TAHO6/CD21 expression pattern as assessed byimmunohistochemistry in tonsil samples, a lymphoid organ where B cellsdevelop, the molecules are excellent targets for therapy of tumors inmammals, including B-cell associated cancers, such as lymphomas (i.e.Non-Hodgkin's Lyphoma), leukemias (i.e. chronic lymphocytic leukemia),myelomas (i.e. multiple myeloma) and other cancers of hematopoieticcells. Further, in light of the TAHO6/CD21 expression pattern asassessed by immunohistochemistry in FFPE samples of DLBCL and B-celllymphomas and frozen samples of DLBCL, B-cell lymphomas and NHL,TAHO25/CD19 may be an excellent target for therapy of tumors in mammals,including B-cell associated cancers, such as lymphomas (i.e.Non-Hodgkin's Lyphoma and diffuse large B-cell lymphoma), leukemias(i.e. chronic lymphocytic leukemia), myelomas (i.e. multiple myeloma)and other cancers of hematopoietic cells that are expressing low amountsof CD21 (CD21-low) or are CD21 negative (CD21−). Specifically, in lightof inhibition of internalization of CD19 in CD21 expressing cells (SeeExample 15), anti-CD19 ADCs which require internalization for efficacy(i.e. anti-CD19-SMCC-DM1) may be more effective in CD21-low orCD-21-cells. Even further, CD21 and CD19 IHC analysis on prospectivepatient tumor samples to determine whether CD21 is absent from amajority of CD19+ cells in those tumor samples may be helpful in patientselection for anti-CD19-SMCC-DM1 therapies.

Example 14 Flow Cytometry

To determine the expression of TAHO molecules, FACS analysis isperformed using a variety of cells, including normal cells and diseasedcells, such as chronic lymphocytic leukemia (CLL) cells.

A. Normal Cells:

For tonsil B cell subtypes, the fresh tonsil is minced in cold HBSS andpassed through a 70 um cell strainer. Cells are washed once and counted.CD19+B cells are enriched using the AutoMACS (Miltenyi). Briefly, tonsilcells are blocked with human IgG, incubated with anti-CD19 microbeads,and washed prior to positive selection over the AutoMACS. A fraction ofCD19+B cells are saved for flow cytometric analysis of plasma cells.Remaining CD19+ cells are stained with FITC-CD77, PE-IgD, and APC-CD38for sorting of B-cell subpopulations. CD19+ enrichment is analyzed usingPE-Cy5-CD19, and purity ranged from 94-98% CD19+. Tonsil Bsubpopulations are sorted on the MoFlo by Michael Hamilton at flow rate18,000-20,000 cells/second. Follicular mantle cells are collected as theIgD+/CD38-fraction, memory B cells were IgD−/CD38−, centrocytes wereIgD−/CD38+/CD77−, and centroblasts were IgD−/CD38+/CD77+. Cells areeither stored in 50% serum overnight, or stained and fixed with 2%paraformaldehyde. For plasma cell analysis, total tonsil B cells arestained with CD138-PE, CD20-FITC, and biotinylated antibody to thetarget of interest detected with streptavidin-PE-Cy5. Tonsil Bsubpopulations are stained with biotinylated antibody to the target ofinterest, detected with streptavidin-PE-Cy5. Flow analysis is done onthe BD FACSCaliber, and data was further analyzed using FlowJo softwarev 4.5.2 (TreeStar). Biotin-conjugated antibodies which are commerciallyavailable such as may be used in the flow cytometry.

Accordingly, the TAHO expression pattern on tonsil-B subtypes asassessed by FACS, may indicate that the TAHO polypeptides are excellenttargets for therapy of tumors in mammals, including B-cell associatedcancers, such as lymphomas (i.e. Non-Hodgkin's Lyphoma), leukemias (i.e.chronic lymphocytic leukemia), myelomas (i.e. multiple myeloma) andother cancers of hematopoietic cells.

B. CLL Cells:

The following purified or fluorochrome-conjugated mAbs are used for flowcytometry of CLL samples: CD5-PE, CD19-PerCP Cy5.5, CD20-FITC, CD20-APC(commercially available from BD Pharmingen). Further, commerciallyavailable biotinylated antibodies against the TAHO molecules may be usedfor the flow cytometry. The CD5, CD19 and CD20 antibodies are used togate on CLL cells and PI staining is performed to check the cellviability.

Cells (10⁶ cells in 100 l volume) are first incubated with 1 g of eachCD5, CD19 and CD20 antibodies and 10 g each of human and mouse gammaglobulin (Jackson ImmunoResearch Laboratories, West Grove, Pa.) to blockthe nonspecific binding, then incubated with optimal concentrations ofmAbs for 30 minutes in the dark at 4° C. When biotinylated antibodiesare used, streptavidin-PE or streptavidin-APC (Jackson ImmunoResearchLaboratories) are then added according to manufacture's instructions.Flow cytometry is performed on a FACS calibur (BD Biosciences, San Jose,Calif.). Forward scatter (FSC) and side scatter (SSC) signals arerecorded in linear mode, fluorescence signals in logarithmic mode. Deadcells and debris are gated out using scatter properties of the cells.Data are analysed using CellQuest Pro software (BD Biosciences) andFlowJo (Tree Star Inc.).

The expression pattern on CLL samples may be performed using monoclonalantibody specific to the TAHO polypeptide of interest.

Accordingly, the TAHO expression pattern on chronic lymphocytic leukemia(CLL) samples as assessed by FACS, may indicate that the TAHO moleculesare excellent targets for therapy of tumors in mammals, including B-cellassociated cancers, such as lymphomas (i.e. Non-Hodgkin's Lyphoma),leukemias (i.e. chronic lymphocytic leukemia), myelomas (i.e. multiplemyeloma) and other cancers of hematopoietic cells.

Example 15 TAHO Internalization

A. Internalization

Internalization of the TAHO antibodies into B-cell lines was assessed inRaji, Ramos, Daudi and other B cell lines, including ARH77, SuDHL4,U698M, huB, BJAB, DoHH2 and Namalwa cell lines. Internalization of theTAHO antibodies was also examined in B-cell lines expressing TAHOpolypeptides.

For further analysis of internalization of anti-TAHO25 (CD19) antibody,a Ramos B-cell line expressing a one amino-acid variant of TAHO6(DNA225875; CD21), herein referred to as TAHO41 (DNA548572; CD21variant) was used (See Example 11 for generation of CD21-expressingRamos Clones).

One ready-to-split 15 cm dish of B-cells (˜50×10⁶ cells) (untransfectedor transfected as described above) with cells for use in up to 20reactions was used. The cells were below passage 25 (less than 8 weeksold) and growing healthily without any mycoplasma.

In a loosely-capped 15 ml Falcon tube add 1 μg/ml mouse anti-TAHOantibody was added to 2.5×10⁶ cells in 2 ml normal growth medium (e.g.RPMI/10% FBS/1% glutamine) containing 1:10 human IgG FcR block (MiltenyiMACS kit, dialyzed to remove azide), 1% pen/strep, 5 μM pepstatin A, 10μg/ml leupeptin (lysosomal protease inhibitors) and 25 μg/mlAlexa488-transferrin (which labeled the recycling pathway and indicatedwhich cells were alive; alternatively Ax488 dextran fluid phase markermay be used to mark all pathways) for 24 hours in a 37° C. 5% CO₂incubator. For quickly-internalizing antibodies, time-points every 5minutes were taken. For time-points taken less than 3 hours, 1 mlcomplete carbonate-free medium (Gibco 18045-088+10% FBS, 1% glutamine,1% pen/strep, 10 mM Hepes pH 7.4) was used and the reactions wereperformed in a 37° C. waterbath instead of the CO₂ incubator.

After completion of the time course as indicated, the cells werecollected by centrifugation (1500 rpm 4° C. for 5 minutes in G6-SR or2500 rpm 3 minutes in 4° C. benchtop eppendorf centrifuge), washed oncein 1.5 ml carbonate free medium (in Eppendorfs) or 10 ml medium for 15ml Falcon tubes. The cells were subjected to a second centrifugation andresuspended in 0.5 ml 3% paraformaldehyde (EMS) in PBS for 20 minutes atroom temp to allow fixation of the cells.

All following steps are followed by a collection of the cells viacentrifugation as above. Cells were washed in PBS and then quenched for10 minutes in 0.5 ml 50 mM NH₄Cl (Sigma) in PBS and permeabalized with0.5 ml 0.1% Triton-X-100 in PBS for 4 minutes during a 4 minutecentrifugation spin. Cells were washed in PBS and subjected tocentrifugation. 1 μg/ml Cy3-anti mouse (or anti-species 1° antibody) wasadded to detect uptake of the antibody in 200 μl complete carbonate freemedium for 20 minutes at room temperature. Cells were washed twice incarbonate free medium and resuspended in 25 μl carbonate free medium andthe cells were allowed to settle as a drop onto one well of apolylysine-coated 8-well LabtekII slide for at least one hour (orovernight in fridge). Any non-bound cells were aspirated and the slideswere mounted with one drop per well of DAPI-containing Vectashield undera 50×24 mm coverslip. The cells were examined under 100× objective forinternalization of the antibodies.

B. FACS of B Cell Lines

For analysis of surface expression of CD19 and CD21 in the B cellsdescribed above, 1 million cells per tube of the indicated cell lineswere incubated with 2 μg/ml anti-CD19 (Biomeda catalog #CB-19, cloneB496, isotype IgG1) or anti-CD21 (ATCC catalog #HB-135, clone THB5 (alsoreferred herein as HB-5), isotype IgG2a) in carbonate-free media(contains 10% FBS, 1% glutamine, 10 mM Hepes, 1% pen/strep) with 1:100FcR block (Miltneyi) on ice for 30 min, then washed twice incarbonate-free media to remove unbound antibody. The bound antibodieswere detected with PE-labeled rat anti-mouse monoclonal IgG1 (for CD19,Benton Dickinson #340270) or IgG2a+b (for CD21, Benton Dickinson#340269) by incubation for 30 minutes on ice, followed by washing twiceand resuspended in 500 μl PBS containing 3% FBS. Propidium iodide wasadded to select out dead cells prior to FACS analysis of the live onesusing an Epics XL machine. Binding was measured by Mean FluorescentIntensity (MFI) (FIG. 9).

Summary

(1) TAHO25/CD19 (as detected using anti-CD19 antibody Biomeda CB-19) wasinternalized within 20 minutes in Ramos and Daudi cells, arriving inlysosomes by 1 hour. In Raji cells and ARH77 cells, TAHO25/CD19internalization was not detectable in 20 hours. Similarly, in Rajicells, TAHO25/CD19 internalization (as detected using anti-CD19antibodies BU12 (AnCell), FMC63 (B19, Chemicon), HD37 (B4, Chemicon),J4.119 (1313, Immunotech) or 1BB250 (US Biologicals)) was not detectablein 3 hours. In DoHH2 and Namalwa cells, internalization was detected by1 hour with the internalization of CD19 (as detected using anti-CD19antibody Biomeda CB-19) in Namalwa cells colocalizing with lysosomes.

TAHO25/CD19 (as detected using anti-CD19 antibody Biomeda Catalog#CB-19, clone B496) internalization was not significantly detectable in3 hours in Ramos cells expressing TAHO41 (CD21 variant), CD21 RamosClone 1. CD19 was also not significantly internalized in Raji and ARH77cells (cells which express CD21 endogenously; See FIG. 9) in 3 hours.Some internalization of TAHO25/CD19 was detectable in 3 hours in CD21Ramos Clone 3, but the amount of internalization was less that observedin 3 hours in untransfected Ramos cells (which do not endogenouslyexpress CD21 (FIG. 9). TAHO25/CD19 was internalized in 3 hours in Ramoscells (not transfected with CD21 variant and not endogenously expressingCD21 (See FIG. 9). Increased inhibition of internalization of CD19antibody in the Ramos expressing CD21 variant correlated with increasedCD21 variant expression in Ramos cells (Ramos Clone 1 and Ramos Clone 3;See FIG. 9). Specifically, in 3 hours, Ramos Clone 3, expressing lessCD21 variant than Ramos Clone 1, inhibited CD19 internalization lessthan Ramos Clone 1.

From FACS analysis, Ramos and DoHH2 cells did not express detectablelevels of CD21 (See FIG. 9), Namalwa, Daudi, ARH77 and Ramos Clone 3cells expressed some level of CD21 (See FIG. 9), and Raji and RamosClone 1 cells expressed significant levels of CD21 (See FIG. 9).Expression of CD21 on ARH77 cells is dependent on the age of the cellsand time lapsing between the last split and the FACS analysis, due toCD21 shedding (Frémeaux-Bacchi et al., International Immunology, 10(10):1459-1466 (1998). Cells that were split the day before being analyzedvia FACS showed levels of CD21 expression closer to Raji cells than toDaudi cells (Data not shown).

(2) Significant TAHO6/CR2/CD21 (as detected using anti-CR2 antibody ATCCHB-135) internalization was not detectable in Raji, ARH77, Namalwa orDaudi cells in 20 hours, irrespective of CD21 expression level.Significant TAHO6/CR2/CD21 internalization was also not detected inRamos and DoHH2 cells, which do not express detectable levels of CD21,in 20 hours.

Accordingly, in light of CD19/TAHO25 and CD21/TAHO6 internalization dataon B-cell lines as assessed by immunofluorescence using respectiveanti-TAHO antibodies, the molecules are excellent targets for therapy oftumors in mammals, including B-cell associated cancers, such aslymphomas (i.e. Non-Hodgkin's Lyphoma), leukemias (i.e. chroniclymphocytic leukemia), myelomas (i.e. multiple myeloma) and othercancers of hematopoietic cells.

Further, the non-internalizing CD21/TAHO6 molecule may be an excellenttarget for antibody therapies that do not require internalization, i.e.naked anti-CD21 antibodies, and anti-CD21 antibody ADCs that do notrequire internalization for drug release (e.g. anti-CD21-SPP-DM1).

Further, anti-TAHO25/CD19 ADCs which require internalization for drugrelease (e.g. anti-CD19-SMCC-DM1) may be particularly effective inCD21-negative or CD21-low and CD19-positive B-cell associated cancers,such as lymphomas (i.e. Non-Hodgkin's Lyphoma, diffuse large B-celllymphoma), leukemias (i.e. chronic lymphocytic leukemia), myelomas (i.e.multiple myeloma) and other cancers of hematopoietic cells whileanti-TAHO25/CD19 ADCs which do not require internalization for drugrelease (e.g. anti-CD19-SPP-DM1) may be effective in CD19+ lymphomas,irrespective of CD21 expression. In light of the IHC data in Example 13,CD21 and CD19 IHC analysis on prospective patient tumor samples todetermine whether CD21 is absent from a majority of CD19+ cells in thosetumor samples may be helpful in patient selection for anti-CD19-SMCC-DM1therapies.

1. Isolated nucleic acid having a nucleotide sequence that has at least95% nucleic acid sequence identity to: (a) a DNA molecule encoding theamino acid sequence selected from the group consisting of the amino acidsequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG.6 (SEQ ID NO: 6); (b) a DNA molecule encoding the amino acid sequenceselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6),lacking its associated signal peptide; (c) a DNA molecule encoding anextracellular domain of the polypeptide having the amino acid selectedfrom the group consisting of the amino acid sequence shown in FIG. 2(SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), withits associated signal peptide; (d) a DNA molecule encoding anextracellular domain of the polypeptide having the amino acid selectedfrom the group consisting of the amino acid sequence shown in FIG. 2(SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lackingits associated signal peptide; (e) the nucleotide sequence selected fromthe group consisting of the nucleotide sequence shown in FIG. 1 (SEQ IDNO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); (f) thefull-length coding region of the nucleotide sequence selected from thegroup consisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or (g) thecomplement of (a), (b), (c), (d), (e) or (f).
 2. Isolated nucleic acidhaving: (a) a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6);(b) a nucleotide sequence that encodes the amino acid sequence selectedfrom the group consisting of the amino acid sequence shown in FIG. 2(SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lackingits associated signal peptide; (c) a nucleotide sequence that encodes anextracellular domain of the polypeptide having the amino acid selectedfrom the group consisting of the amino acid sequence shown in FIG. 2(SEQ ID NO: 2), FIG. (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), with itsassociated signal peptide; (d) a nucleotide sequence that encodes anextracellular domain of the polypeptide having the amino acid selectedfrom the group consisting of the amino acid sequence shown in FIG. 2(SEQ ID NO: 2), FIG. (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lackingits associated signal peptide; (e) the nucleotide sequence selected fromthe group consisting of the nucleotide sequence shown in FIG. 1 (SEQ IDNO: 1) and FIG. 3 (SEQ ID NO:3); (f) the full-length coding region ofthe nucleotide sequence selected from the group consisting of thenucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3)and FIG. 5 (SEQ ID NO: 5); or (g) the complement of (a), (b), (c), (d),(e) or (f).
 3. Isolated nucleic acid that hybridizes to: (a) a nucleicacid that encodes the amino acid sequence selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2),FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6); (b) a nucleic acid thatencodes the amino acid sequence selected from the group consisting ofthe amino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ IDNO: 4) and FIG. 6 (SEQ ID NO: 6), lacking its associated signal peptide;(c) a nucleic acid that encodes an extracellular domain of thepolypeptide having the amino acid sequence selected from the groupconsisting of the amino acid sequence shown FIG. 2 (SEQ ID NO: 2), FIG.4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), with its associated signalpeptide; (d) a nucleic acid that encodes an extracellular domain of thepolypeptide having the amino acid sequence selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2),FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lacking its associatedsignal peptide; (e) the nucleotide sequence selected from the groupconsisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1),FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); (f) the full-lengthcoding region of the nucleotide sequence selected from the groupconsisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1),FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or (g) the complement of(a), (b), (c), (d), (e) or (f).
 4. The nucleic acid of claim 3, whereinthe hybridization occurs under stringent conditions.
 5. The nucleic acidof claim 3, which is at least about 5 nucleotides in length.
 6. Anexpression vector comprising the nucleic acid of claim 1, 2 or
 3. 7. Theexpression vector of claim 6, wherein said nucleic acid is operablylinked to control sequences recognized by a host cell transformed withthe vector.
 8. A host cell comprising the expression vector of claim 7.9. The host cell of claim 8 which is a CHO cell, an E. coli cell or ayeast cell.
 10. A process for producing a polypeptide comprisingculturing the host cell of claim 9 under conditions suitable forexpression of said polypeptide and recovering said polypeptide from thecell culture.
 11. An isolated polypeptide having at least 95% amino acidsequence identity to: (a) the polypeptide having the amino acid sequenceselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6);(b) the polypeptide having the amino acid sequence selected from thegroup consisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lacking itsassociated signal peptide; (c) an extracellular domain of thepolypeptide having the amino acid sequence selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2),FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), with its associatedsignal peptide; (d) an extracellular domain of the polypeptide havingthe amino acid sequence selected from the group consisting of the aminoacid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) andFIG. 6 (SEQ ID NO: 6) lacking its associated signal peptide; (e) apolypeptide encoded by the nucleotide sequence selected from the groupconsisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1),FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or (f) a polypeptideencoded by the full-length coding region of the nucleotide sequenceselected from the group consisting of the nucleotide sequence shown inFIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5).12. An isolated polypeptide having: (a) the amino acid sequence selectedfrom the group consisting of the amino acid sequence shown in FIG. 2(SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6); (b) theamino acid sequence selected from the group consisting of the amino acidsequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG.6 (SEQ ID NO: 6), lacking its associated signal peptide sequence; (c) anamino acid sequence of an extracellular domain of the polypeptideselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6),with its associated signal peptide sequence; (d) an amino acid sequenceof an extracellular domain of the polypeptide selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2),FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lacking its associatedsignal peptide sequence; (e) an amino acid sequence encoded by thenucleotide sequence selected from the group consisting of the nucleotidesequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO: 3) and FIG.5 (SEQ ID NO: 5); or (f) an amino acid sequence encoded by thefull-length coding region of the nucleotide sequence selected from thegroup consisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1), FIG. 3 (SEQ ID NO: 3) and FIG. 5 (SEQ ID NO: 5).
 13. A chimericpolypeptide comprising the polypeptide of claim 52 or 53 fused to aheterologous polypeptide.
 14. The chimeric polypeptide of claim 54,wherein said heterologous polypeptide is an epitope tag sequence or anFc region of an immunoglobulin.
 15. An isolated antibody that binds to apolypeptide having at least 95% amino acid sequence identity to: (a) thepolypeptide having the amino acid sequence selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2),FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6); (b) the polypeptideselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6),lacking its associated signal peptide; (c) an extracellular domain ofthe polypeptide having the amino acid sequence selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2),FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), with its associatedsignal peptide; (d) an extracellular domain of the polypeptide havingthe amino acid sequence selected from the group consisting of the aminoacid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) andFIG. 6 (SEQ ID NO: 6), lacking its associated signal peptide; (e) apolypeptide encoded by the nucleotide sequence selected from the groupconsisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1),FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or (f) a polypeptideencoded by the full-length coding region of the nucleotide sequenceselected from the group consisting of the nucleotide sequence shown inFIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5).16. An isolated antibody that binds to a polypeptide having: (a) theamino acid sequence selected from the group consisting of the amino acidsequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG.6 (SEQ ID NO: 6); (b) the amino acid sequence selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2),FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lacking its associatedsignal peptide sequence; (c) an amino acid sequence of an extracellulardomain of the polypeptide selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO:4) and FIG. 6 (SEQ ID NO: 6), with its associated signal peptidesequence; (d) an amino acid sequence of an extracellular domain of thepolypeptide selected from the group consisting of the amino acidsequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG.6 (SEQ ID NO: 6), lacking its associated signal peptide sequence; (e) anamino acid sequence encoded by the nucleotide sequence selected from thegroup consisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1), FIG. 3 (SEQ ID NO: 3) and FIG. 5 (SEQ ID NO: 5); or (f) an aminoacid sequence encoded by the full-length coding region of the nucleotidesequence selected from the group consisting of the nucleotide sequenceshown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO: 3) and FIG. 5 (SEQ IDNO: 5).
 17. The antibody of claim 15 or 16 which is a monoclonalantibody.
 18. The antibody of claim 15 or 16 which is an antibodyfragment.
 19. The antibody of claim 15 or 16 which is a chimeric or ahumanized antibody.
 20. The antibody of claim 15 or 16 which isconjugated to a growth inhibitory agent.
 21. The antibody of claim 15 or16 which is conjugated to a cytotoxic agent.
 22. The antibody of claim21, wherein the cytotoxic agent is selected from the group consisting oftoxins, antibiotics, radioactive isotopes and nucleolytic enzymes. 23.The antibody of claim 22, wherein the toxin is selected from the groupconsisting of maytansinoid, auristatin peptide and calicheamicin. 24.The antibody of claim 15 or 16 which induces death of a cell to which itbinds.
 25. The antibody of claim 15 or 16 which is detectably labeled.26. An isolated nucleic acid having a nucleotide sequence that encodesthe antibody of claim 15 or
 16. 27. An expression vector comprising thenucleic acid of claim 26 operably linked to control sequences recognizedby a host cell transformed with the vector.
 28. A host cell comprisingthe expression vector of claim
 27. 29. The host cell of claim 28 whichis a CHO cell, an E. coli cell or a yeast cell.
 30. A process forproducing an antibody comprising culturing the host cell of claim 29under conditions suitable for expression of said antibody and recoveringsaid antibody from the cell culture.
 31. An isolated oligopeptide thatbinds to a polypeptide having at least 95% amino acid sequence identityto: (a) the polypeptide having the amino acid sequence selected from thegroup consisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6); (b) the polypeptidehaving the amino acid sequence selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO:4) and FIG. 6 (SEQ ID NO: 6), lacking its associated signal peptide; (c)an extracellular domain of the polypeptide having the amino acidsequence selected from the group consisting of the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ IDNO: 6), with its associated signal peptide; (d) an extracellular domainof the polypeptide having the amino acid sequence selected from thegroup consisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lacking itsassociated signal peptide; (e) a polypeptide encoded by the nucleotidesequence selected from the group consisting of the nucleotide sequenceshown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ IDNO: 5); or (f) a polypeptide encoded by the full-length coding region ofthe nucleotide sequence selected from the group consisting of thenucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3)and FIG. 5 (SEQ ID NO: 5).
 32. An isolated oligopeptide that binds to apolypeptide having: (a) the amino acid sequence selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2),FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6); (b) the amino acidsequence selected from the group consisting of the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ IDNO: 6), lacking its associated signal peptide sequence; (c) an aminoacid sequence of an extracellular domain of the polypeptide selectedfrom the group consisting of the amino acid sequence shown in FIG. 2(SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), withits associated signal peptide sequence; (d) an amino acid sequence of anextracellular domain of the polypeptide selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2),FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lacking its associatedsignal peptide sequence; (e) an amino acid sequence encoded by thenucleotide sequence selected from the group consisting of the nucleotidesequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5(SEQ ID NO: 5); or (f) an amino acid sequence encoded by the full-lengthcoding region of the nucleotide sequence selected from the groupconsisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1),FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5).
 33. The oligopeptide ofclaim 31 or 32 which is conjugated to a growth inhibitory agent.
 34. Theoligopeptide of claim 31 or 32 which is conjugated to a cytotoxic agent.35. The oligopeptide of claim 34, wherein the cytotoxic agent isselected from the group consisting of toxins, antibiotics, radioactiveisotopes and nucleolytic enzymes.
 36. The oligopeptide of claim 35,wherein the toxin is selected from the group consisting of maytansinoid,auristatin peptide and calicheamicin.
 37. The oligopeptide of claim 31or 32 which induces death of a cell to which it binds.
 38. Theoligopeptide of claim 31 or 32 which is detectably labeled.
 39. A TAHObinding organic molecule that binds to a polypeptide having at least 95%amino acid sequence identity to: (a) the polypeptide having the aminoacid sequence selected from the group consisting of the amino acidsequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG.6 (SEQ ID NO: 6); (b) the polypeptide having the amino acid sequenceselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6),lacking its associated signal peptide; (c) an extracellular domain ofthe polypeptide having the amino acid sequence selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2),FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), with its associatedsignal peptide; (d) an extracellular domain of the polypeptide havingthe amino acid sequence selected from the group consisting of the aminoacid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) andFIG. 6 (SEQ ID NO: 6), lacking its associated signal peptide; (e) apolypeptide encoded by the nucleotide sequence selected from the groupconsisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1),FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or (f) a polypeptideencoded by the full-length coding region of the nucleotide sequenceselected from the group consisting of the nucleotide sequence shown inFIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5).40. The organic molecule of claim 39 that binds to a polypeptide having:(a) the amino acid sequence selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO:4) and FIG. 6 (SEQ ID NO: 6); (b) the amino acid sequence selected fromthe group consisting of the amino acid sequence shown in FIG. 2 (SEQ IDNO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lacking itsassociated signal peptide sequence; (c) an amino acid sequence of anextracellular domain of the polypeptide selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2),FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), with its associatedsignal peptide sequence; (d) an amino acid sequence of an extracellulardomain of the polypeptide selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO:4) and FIG. 6 (SEQ ID NO: 6), lacking its associated signal peptidesequence; (e) an amino acid sequence encoded by the nucleotide sequenceselected from the group consisting of the nucleotide sequence shown inFIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5);or (f) an amino acid sequence encoded by the full-length coding regionof the nucleotide sequence selected from the group consisting of thenucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3)and FIG. 5 (SEQ ID NO: 5).
 41. The organic molecule of claim 39 or 40which is conjugated to a growth inhibitory agent.
 42. The organicmolecule of claim 39 or 40 which is conjugated to a cytotoxic agent. 43.The organic molecule of claim 42, wherein the cytotoxic agent isselected from the group consisting of toxins, antibiotics, radioactiveisotopes and nucleolytic enzymes.
 44. The organic molecule of claim 43,wherein the toxin is selected from the group consisting of maytansinoid,auristatin peptide and calicheamicin.
 45. The organic molecule of claim39 or 40 which induces death of a cell to which it binds.
 46. Theorganic molecule of claim 39 or 40 which is detectably labeled.
 47. Acomposition of matter comprising: (a) the polypeptide of claim 1; (b)the polypeptide of claim 2; (c) the antibody of claim 15; (d) theantibody of claim 16; (e) the oligopeptide of claim 31; (f) theoligopeptide of claim 32; (g) the TAHO binding organic molecule of claim39; or (h) the TAHO binding organic molecule of claim 40; in combinationwith a carrier.
 48. The composition of matter of claim 47, wherein saidcarrier is a pharmaceutically acceptable carrier.
 49. An article ofmanufacture comprising: (a) a container; and (b) the composition ofmatter of claim 47 contained within said container.
 50. The article ofmanufacture of claim 49 further comprising a label affixed to saidcontainer, or a package insert included with said container, referringto the use of said composition of matter for the therapeutic treatmentof or the diagnostic detection of a cancer.
 51. A method of inhibitingthe growth of a cell that expresses a protein having at least 95% aminoacid sequence identity to: (a) the polypeptide having the amino acidsequence selected from the group consisting of the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ IDNO: 6); (b) the polypeptide having the amino acid sequence selected fromthe group consisting of the amino acid sequence shown in FIG. 2 (SEQ IDNO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lacking itsassociated signal peptide; (c) an extracellular domain of thepolypeptide having the amino acid sequence selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2),FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), with its associatedsignal peptide; (d) an extracellular domain of the polypeptide havingthe amino acid sequence selected from the group consisting of the aminoacid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) andFIG. 6 (SEQ ID NO: 6), lacking its associated signal peptide; (e) apolypeptide encoded by the nucleotide sequence selected from the groupconsisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1),FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or (f) a polypeptideencoded by the full-length coding region of the nucleotide sequenceselected from the group consisting of the nucleotide sequence shown inFIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5),said method comprising contacting said cell with an antibody,oligopeptide or organic molecule that binds to said protein, the bindingof said antibody, oligopeptide or organic molecule to said proteinthereby causing an inhibition of growth of said cell.
 52. The method ofclaim 51, wherein said antibody is a monoclonal antibody.
 53. The methodof claim 51, wherein said antibody is an antibody fragment.
 54. Themethod of claim 51, wherein said antibody is a chimeric or a humanizedantibody.
 55. The method of claim 51, wherein said antibody,oligopeptide or organic molecule is conjugated to a growth inhibitoryagent.
 56. The method of claim 51, wherein said antibody, oligopeptideor organic molecule is conjugated to a cytotoxic agent.
 57. The methodof claim 56, wherein said cytotoxic agent is selected from the groupconsisting of toxins, antibiotics, radioactive isotopes and nucleolyticenzymes.
 58. The method of claim 57, wherein the toxin is selected fromthe group consisting of maytansinoid, auristatin peptide andcalicheamicin.
 59. The method of claim 51, wherein said cell is ahematopoietic cell.
 60. The method of claim 59, wherein saidhematopoietic cell is selected from the group consisting of alymphocyte, leukocyte, platelet, erythrocyte and natural killer cell.61. The method of claim 60, wherein said lymphocyte is a B cell or Tcell.
 62. The method of claim 60, wherein said lymphocyte is a cancercell.
 63. The method of claim 62, wherein said cancer cell is furtherexposed to radiation treatment or a chemotherapeutic agent.
 64. Themethod of claim 62, wherein said cancer cell is selected from the groupconsisting of a lymphoma cell, a myeloma cell and a leukemia cell. 65.The method of claim 59, wherein said protein is more abundantlyexpressed by said hematopoietic cell as compared to a non-hematopoieticcell.
 66. The method of claim 51 which causes the death of said cell.67. The method of claim 51, wherein said protein has: (a) the amino acidsequence selected from the group consisting of the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ IDNO: 6); (b) the amino acid sequence selected from the group consistingof the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQID NO: 4) and FIG. 6 (SEQ ID NO: 6), lacking its associated signalpeptide sequence; (c) an amino acid sequence of an extracellular domainof the polypeptide selected from the group consisting of the amino acidsequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG.6 (SEQ ID NO: 6), with its associated signal peptide sequence; (d) anamino acid sequence of an extracellular domain of the polypeptideselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6),lacking its associated signal peptide sequence; (e) an amino acidsequence encoded by the nucleotide sequence selected from the groupconsisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1),FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or (f) an amino acidsequence encoded by the full-length coding region of the nucleotidesequence selected from the group consisting of the nucleotide sequenceshown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ IDNO: 5).
 68. A method of therapeutically treating a mammal having acancerous tumor comprising cells that express a protein having at least95% amino acid sequence identity to: (a) the polypeptide having theamino acid sequence selected from the group consisting of the amino acidsequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG.6 (SEQ ID NO: 6); (b) the polypeptide having the amino acid sequenceselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6),lacking its associated signal peptide; (c) an extracellular domain ofthe polypeptide having the amino acid sequence selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2),FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), with its associatedsignal peptide; (d) an extracellular domain of the polypeptide havingthe amino acid sequence selected from the group consisting of the aminoacid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) andFIG. 6 (SEQ ID NO: 6), lacking its associated signal peptide; (e) apolypeptide encoded by the nucleotide sequence selected from the groupconsisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1),FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or (f) a polypeptideencoded by the full-length coding region of the nucleotide sequenceselected from the group consisting of the nucleotide sequence shown inFIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5),said method comprising administering to said mammal a therapeuticallyeffective amount of an antibody, oligopeptide or organic molecule thatbinds to said protein, thereby effectively treating said mammal.
 69. Themethod of claim 68, wherein said antibody is a monoclonal antibody. 70.The method of claim 68, wherein said antibody is an antibody fragment.71. The method of claim 68, wherein said antibody is a chimeric or ahumanized antibody.
 72. The method of claim 68, wherein said antibody,oligopeptide or organic molecule is conjugated to a growth inhibitoryagent.
 73. The method of claim 68, wherein said antibody, oligopeptideor organic molecule is conjugated to a cytotoxic agent.
 74. The methodof claim 73, wherein said cytotoxic agent is selected from the groupconsisting of toxins, antibiotics, radioactive isotopes and nucleolyticenzymes.
 75. The method of claim 74, wherein the toxin is selected fromthe group consisting of maytansinoid, C auristatin peptide andcalicheamicin.
 76. The method of claim 68, wherein said tumor is furtherexposed to radiation treatment or a chemotherapeutic agent.
 77. Themethod of claim 68, wherein said tumor is a lymphoma, leukemia ormyeloma tumor.
 78. The method of claim 68, wherein said protein is moreabundantly expressed by a hematopoietic cell as compared to anon-hematopoietic cell of said tumor.
 79. The method of claim 78,wherein said protein is more abundantly expressed by canceroushematopoietic cells of said tumor as compared to normal hematopoieticcells of said tumor.
 80. The method of claim 68, wherein said proteinhas: (a) the amino acid sequence selected from the group consisting ofthe amino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ IDNO: 4) and FIG. 6 (SEQ ID NO: 6); (b) the amino acid sequence selectedfrom the group consisting of the amino acid sequence shown in FIG. 2(SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lackingits associated signal peptide sequence; (c) an amino acid sequence of anextracellular domain of the polypeptide selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2),FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), with its associatedsignal peptide sequence; (d) an amino acid sequence of an extracellulardomain of the polypeptide selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO:4) and FIG. 6 (SEQ ID NO: 6), lacking its associated signal peptidesequence; (e) an amino acid sequence encoded by the nucleotide sequenceselected from the group consisting of the nucleotide sequence shown inFIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5);or (f) an amino acid sequence encoded by the full-length coding regionof the nucleotide sequence selected from the group consisting of thenucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3)and FIG. 5 (SEQ ID NO: 5).
 81. A method of determining the presence of aprotein in a sample suspected of containing said protein, wherein saidprotein has at least 95% amino acid sequence identity to: (a) thepolypeptide having the amino acid sequence selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2),FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6); (b) the polypeptidehaving the amino acid sequence selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO:4) and FIG. 6 (SEQ ID NO: 6), lacking its associated signal peptide; (c)an extracellular domain of the polypeptide having the amino acidsequence selected from the group consisting of the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ IDNO: 6), with its associated signal peptide; (d) an extracellular domainof the polypeptide having the amino acid sequence selected from thegroup consisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lacking itsassociated signal peptide; (e) a polypeptide encoded by the nucleotidesequence selected from the group consisting of the nucleotide sequenceshown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ IDNO: 5); or (f) a polypeptide encoded by the full-length coding region ofthe nucleotide sequence selected from the group consisting of thenucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3)and FIG. 5 (SEQ ID NO: 5), said method comprising exposing said sampleto an antibody, oligopeptide or organic molecule that binds to saidprotein and determining binding of said antibody, oligopeptide ororganic molecule to said protein in said sample, wherein binding of theantibody, oligopeptide or organic molecule to said protein is indicativeof the presence of said protein in said sample.
 82. The method of claim81, wherein said sample comprises a cell suspected of expressing saidprotein.
 83. The method of claim 81, wherein said cell is a cancer cell.84. The method of claim 81, wherein said antibody, oligopeptide ororganic molecule is detectably labeled.
 85. The method of claim 83,wherein said cancer cell is selected from the group consisting oflymphoma, leukemia or myeloma cells.
 86. The method of claim 81, whereinsaid protein is more abundantly expressed by a hematopoietic cell ascompared to a non-hematopoietic cell.
 87. The method of claim 81,wherein said protein is more abundantly expressed by a canceroushematopoietic cell as compared to normal hematopoietic cell.
 88. Themethod of claim 81, wherein said protein has: (a) the amino acidsequence selected from the group consisting of the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ IDNO: 6); (b) the amino acid sequence selected from the group consistingof the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQID NO: 4) and FIG. 6 (SEQ ID NO: 6), lacking its associated signalpeptide sequence; (c) an amino acid sequence of an extracellular domainof the polypeptide selected from the group consisting of the amino acidsequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG.6 (SEQ ID NO: 6), with its associated signal peptide sequence; (d) anamino acid sequence of an extracellular domain of the polypeptideselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6),lacking its associated signal peptide sequence; (e) an amino acidsequence encoded by the nucleotide sequence selected from the groupconsisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1),FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or (f) an amino acidsequence encoded by the full-length coding region of the nucleotidesequence selected from the group consisting of the nucleotide sequenceshown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ IDNO: 5).
 89. A method for treating or preventing a cell proliferativedisorder associated with increased expression or activity of a proteinhaving at least 95% amino acid sequence identity to: (a) the polypeptidehaving the amino acid sequence selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO:4) and FIG. 6 (SEQ ID NO: 6); (b) the polypeptide having the amino acidsequence selected from the group consisting of the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ IDNO: 6), lacking its associated signal peptide; (c) an extracellulardomain of the polypeptide having the amino acid sequence selected fromthe group consisting of the amino acid sequence shown in FIG. 2 (SEQ IDNO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6) with itsassociated signal peptide; (d) an extracellular domain of thepolypeptide having the amino acid sequence selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2),FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lacking its associatedsignal peptide; (e) a polypeptide encoded by the nucleotide sequenceselected from the group consisting of the nucleotide sequence shown inFIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5);or (f) a polypeptide encoded by the full-length coding region of thenucleotide sequence selected from the group consisting of the nucleotidesequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5(SEQ ID NO: 5), said method comprising administering to a subject inneed of such treatment an effective amount of an antagonist of saidprotein, thereby effectively treating or preventing said cellproliferative disorder.
 90. The method of claim 89, wherein said cellproliferative disorder is cancer.
 91. The method of claim 89, whereinsaid antagonist is an anti-TAHO polypeptide antibody, TAHO bindingoligopeptide, TAHO binding organic molecule or antisenseoligonucleotide.
 92. The method of claim 89, wherein said antibody is amonoclonal antibody.
 93. The method of claim 89, wherein said antibodyis an antibody fragment.
 94. The method of claim 89, wherein saidantibody is a chimeric or a humanized antibody.
 95. The method of claim89, wherein said antibody, oligopeptide or organic molecule isconjugated to a growth inhibitory agent.
 96. The method of claim 89,wherein said antibody, oligopeptide or organic molecule is conjugated toa cytotoxic agent.
 97. The method of claim 96, wherein said cytotoxicagent is selected from the group consisting of toxins, antibiotics,radioactive isotopes and nucleolytic enzymes.
 98. The method of claim97, wherein the toxin is selected from the group consisting ofmaytansinoid, auristatin peptide and calicheamicin.
 99. The method ofclaim 90, wherein said cancer is further exposed to radiation treatmentor a chemotherapeutic agent.
 100. The method of claim 90, wherein saidcancer is a lymphoma, leukemia or myeloma.
 101. The method of claim 89,wherein said protein is more abundantly expressed by a hematopoieticcell as compared to a non-hematopoietic cell.
 102. The method of claim89, wherein said protein is more abundantly expressed by canceroushematopoietic cells as compared to normal hematopoietic cells.
 103. Amethod of binding an antibody, oligopeptide or organic molecule to acell that expresses a protein having at least 95% amino acid sequenceidentity to: (a) the polypeptide having the amino acid sequence selectedfrom the group consisting of the amino acid sequence shown in FIG. 2(SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6); (b) thepolypeptide having the amino acid sequence selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2),FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lacking its associatedsignal peptide; (c) an extracellular domain of the polypeptide havingthe amino acid sequence selected from the group consisting of the aminoacid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) andFIG. 6 (SEQ ID NO: 6), with its associated signal peptide; (d) anextracellular domain of the polypeptide having the amino acid sequenceselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6),lacking its associated signal peptide; (e) a polypeptide encoded by thenucleotide sequence selected from the group consisting of the nucleotidesequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5(SEQ ID NO: 5); or (f) a polypeptide encoded by the full-length codingregion of the nucleotide sequence selected from the group consisting ofthe nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ IDNO:3) and FIG. 5 (SEQ ID NO: 5), said method comprising contacting saidcell with an antibody, oligopeptide or organic molecule that binds tosaid protein and allowing the binding of the antibody, oligopeptide ororganic molecule to said protein to occur, thereby binding saidantibody, oligopeptide or organic molecule to said cell.
 104. The methodof claim 103, wherein said antibody is a monoclonal antibody.
 105. Themethod of claim 103, wherein said antibody is an antibody fragment. 106.The method of claim 103, wherein said antibody is a chimeric or ahumanized antibody.
 107. The method of claim 103, wherein said antibody,oligopeptide or organic molecule is conjugated to a growth inhibitoryagent.
 108. The method of claim 103, wherein said antibody, oligopeptideor organic molecule is conjugated to a cytotoxic agent.
 109. The methodof claim 108, wherein said cytotoxic agent is selected from the groupconsisting of toxins, antibiotics, radioactive isotopes and nucleolyticenzymes.
 110. The method of claim 109, wherein the toxin is selectedfrom the group consisting of maytansinoid, auristatin peptide andcalicheamicin.
 111. The method of claim 1103, wherein said cell is ahematopoietic cell.
 112. The method of claim 111, wherein saidhematopoietic cell is a selected from the group consisting of alymphocyte, leukocyte, platelet, erythrocyte and natural killer cell.113. The method of claim 112, wherein said lymphocyte is a B cell or a Tcell.
 114. The method of claim 112, wherein said lymphocyte is a cancercell.
 115. The method of claim 114, wherein said cancer cell is furtherexposed to radiation treatment or a chemotherapeutic agent.
 116. Themethod of claim 114, wherein said cancer cell is selected from the groupconsisting of a leukemia cell, a lymphoma cell and a myeloma cell. 117.The method of claim 111, wherein said protein is more abundantlyexpressed by said hematopoietic cell as compared to a non-hematopoieticcell.
 118. The method of claim 103 which causes the death of said cell.119. The method of claim 103, wherein said protein has: (a) the aminoacid sequence selected from the group consisting of the amino acidsequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG.6 (SEQ ID NO: 6); (b) the amino acid sequence selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2),FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lacking its associatedsignal peptide sequence; (c) an amino acid sequence of an extracellulardomain of the polypeptide selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO:4) and FIG. 6 (SEQ ID NO: 6), with its associated signal peptidesequence; (d) an amino acid sequence of an extracellular domain of thepolypeptide selected from the group consisting of the amino acidsequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG.6 (SEQ ID NO: 6), lacking its associated signal peptide sequence; (e) anamino acid sequence encoded by the nucleotide sequence selected from thegroup consisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or (f) an amino acidsequence encoded by the full-length coding region of the nucleotidesequence selected from the group consisting of the nucleotide sequenceshown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ IDNO: 5).
 120. A method for inhibiting the growth of a cell, wherein thegrowth of said cell is at least in part dependent upon a growthpotentiating effect of a protein having at least 95% amino acid sequenceidentity to: (a) the polypeptide having the amino acid sequence selectedfrom the group consisting of the amino acid sequence shown in FIG. 2(SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6); (b) thepolypeptide having the amino acid sequence selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2),FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lacking its associatedsignal peptide; (c) an extracellular domain of the polypeptide havingthe amino acid sequence selected from the group consisting of the aminoacid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) andFIG. 6 (SEQ ID NO: 6), with its associated signal peptide; (d) anextracellular domain of the polypeptide having the amino acid sequenceselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6),lacking its associated signal peptide; (e) a polypeptide encoded by thenucleotide sequence selected from the group consisting of the nucleotidesequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5(SEQ ID NO: 5), or (f) a polypeptide encoded by the full-length codingregion of the nucleotide sequence selected from the group consisting ofthe nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ IDNO:3) and FIG. 5 (SEQ ID NO: 5), said method comprising contacting saidprotein with an antibody, oligopeptide or organic molecule that binds tosaid protein, there by inhibiting the growth of said cell.
 121. Themethod of claim 120, wherein said cell is a hematopoietic cell.
 122. Themethod of claim 120, wherein said protein is expressed by said cell.123. The method of claim 120, wherein the binding of said antibody,oligopeptide or organic molecule to said protein antagonizes a cellgrowth-potentiating activity of said protein.
 124. The method of claim120, wherein the binding of said antibody, oligopeptide or organicmolecule to said protein induces the death of said cell.
 125. The methodof claim 120, wherein said antibody is a monoclonal antibody.
 126. Themethod of claim 120, wherein said antibody is an antibody fragment. 127.The method of claim 120, wherein said antibody is a chimeric or ahumanized antibody.
 128. The method of claim 120, wherein said antibody,oligopeptide or organic molecule is conjugated to a growth inhibitoryagent.
 129. The method of claim 120, wherein said antibody, oligopeptideor organic molecule is conjugated to a cytotoxic agent.
 130. The methodof claim 129, wherein said cytotoxic agent is selected from the groupconsisting of toxins, antibiotics, radioactive isotopes and nucleolyticenzymes.
 131. The method of claim 130, wherein the toxin is selectedfrom the group consisting of maytansinoid, auristatin peptide andcalicheamicin.
 132. The method of claim 121, wherein said hematopoieticcell is selected from the group consisting of a lymphocyte, leukocyte,platelet, erythrocyte and natural killer cell.
 133. The method of claim132, wherein said lymphocyte is a B cell or T cell.
 134. The method ofclaim 132, wherein said lymphocyte is a cancer cell.
 135. The method ofclaim 134, wherein said cancer cell is selected from the groupconsisting of a lymphoma cell, a myeloma cell and a leukemia cell. 136.The method of claim 121, wherein said protein is more abundantlyexpressed by said hematopoietic cell as compared to a non-hematopoieticcell.
 137. The method of claim 120, wherein said protein has: (a) theamino acid sequence selected from the group consisting of the amino acidsequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG.6 (SEQ ID NO: 6); (b) the amino acid sequence selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2),FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lacking its associatedsignal peptide sequence; (c) an amino acid sequence of an extracellulardomain of the polypeptide selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO:4) and FIG. 6 (SEQ ID NO: 6), with its associated signal peptidesequence; (d) an amino acid sequence of an extracellular domain of thepolypeptide selected from the group consisting of the amino acidsequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG.6 (SEQ ID NO: 6), lacking its associated signal peptide sequence; (e) anamino acid sequence encoded by the nucleotide sequence selected from thegroup consisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or (f) an amino acidsequence encoded by the full-length coding region of the nucleotidesequence selected from the group consisting of the nucleotide sequenceshown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ IDNO: 5).
 138. A method of therapeutically treating a tumor in a mammal,wherein the growth of said tumor is at least in part dependent upon agrowth potentiating effect of a protein having at least 95% amino acidsequence identity to: (a) the polypeptide having the amino acid sequenceselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6);(b) the polypeptide having the amino acid sequence selected from thegroup consisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lacking itsassociated signal peptide; (c) an extracellular domain of thepolypeptide having the amino acid sequence selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2),FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), with its associatedsignal peptide; (d) an extracellular domain of the polypeptide havingthe amino acid sequence selected from the group consisting of the aminoacid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) andFIG. 6 (SEQ ID NO: 6), lacking its associated signal peptide; (e) apolypeptide encoded by the nucleotide sequence selected from the groupconsisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1),FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or (f) a polypeptideencoded by the full-length coding region of the nucleotide sequenceselected from the group consisting of the nucleotide sequence shown inFIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5),said method comprising contacting said protein with an antibody,oligopeptide or organic molecule that binds to said protein, therebyeffectively treating said tumor.
 139. The method of claim 138, whereinsaid protein is expressed by cells of said tumor.
 140. The method ofclaim 138, wherein the binding of said antibody, oligopeptide or organicmolecule to said protein antagonizes a cell growth-potentiating activityof said protein.
 141. The method of claim 138, wherein said antibody isa monoclonal antibody.
 142. The method of claim 138, wherein saidantibody is an antibody fragment.
 143. The method of claim 138, whereinsaid antibody is a chimeric or a humanized antibody.
 144. The method ofclaim 138, wherein said antibody, oligopeptide or organic molecule isconjugated to a growth inhibitory agent.
 145. The method of claim 138,wherein said antibody, oligopeptide or organic molecule is conjugated toa cytotoxic agent.
 146. The method of claim 145, wherein said cytotoxicagent is selected from the group consisting of toxins, antibiotics,radioactive isotopes and nucleolytic enzymes.
 147. The method of claim146, wherein the toxin is selected from the group consisting ofmaytansinoid, auristatin peptide and calicheamicin.
 148. The method ofclaim 138, wherein said tumor is further exposed to radiation treatmentor a chemotherapeutic agent.
 149. The method of claim 138, wherein saidtumor is selected from the group consisting of lymphoma, myeloma andleukemia.
 150. The method of claim 138, wherein said protein is moreabundantly expressed by a hematopoietic cell as compared to anon-hematopoietic cell of said tumor.
 151. The method of claim 1150,wherein said protein is more abundantly expressed by canceroushematopoietic cells of said tumor as compared to normal hematopoieticcells of said tumor.
 152. The method of claim 138, which causes thedeath of said tumor.
 153. The method of claim 138, wherein said proteinhas: (a) the amino acid sequence selected from the group consisting ofthe amino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ IDNO: 4) and FIG. 6 (SEQ ID NO: 6); (b) the amino acid sequence selectedfrom the group consisting of the amino acid sequence shown in FIG. 2(SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lackingits associated signal peptide sequence; (c) an amino acid sequence of anextracellular domain of the polypeptide selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2),FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), with its associatedsignal peptide sequence; (d) an amino acid sequence of an extracellulardomain of the polypeptide selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO:4) and FIG. 6 (SEQ ID NO: 6), lacking its associated signal peptidesequence; (e) an amino acid sequence encoded by the nucleotide sequenceselected from the group consisting of the nucleotide sequence shown inFIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5);or (f) an amino acid sequence encoded by the full-length coding regionof the nucleotide sequence selected from the group consisting of thenucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3)and FIG. 5 (SEQ ID NO: 5).
 154. A method of diagnosing the presence of atumor in a mammal, said method comprising detecting the presence ofcells expressing a polypeptide comprising an amino acid sequence havingat least 95% amino acid sequence identity to: (a) the polypeptide havingthe amino acid sequence selected from the group consisting of the aminoacid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) andFIG. 6 (SEQ ID NO: 6); (b) the polypeptide having the amino acidsequence selected from the group consisting of the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ IDNO: 6), lacking its associated signal peptide; (c) an extracellulardomain of the polypeptide having the amino acid sequence selected fromthe group consisting of the amino acid sequence shown in FIG. 2 (SEQ IDNO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), with itsassociated signal peptide; (d) an extracellular domain of thepolypeptide having the amino acid sequence selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2),FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lacking its associatedsignal peptide; (e) a polypeptide encoded by the nucleotide sequenceselected from the group consisting of the nucleotide sequence shown inFIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5);or (f) a polypeptide encoded by the full-length coding region of thenucleotide sequence selected from the group consisting of the nucleotidesequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5(SEQ ID NO: 5), said method comprising contacting a test sample oftissue cells from said mammal with an antibody, oligopeptide or organicmolecule that binds to said protein and detecting the formation of acomplex between said antibody, oligopeptide or organic molecule, whereinan increased number of cells demonstrating said complex formation, ascompared to a control sample, is indicative of the presence of a tumorin said mammal.
 155. The method of claim 154, wherein said antibody is amonoclonal antibody.
 156. The method of claim 154, wherein said antibodyis an antibody fragment.
 157. The method of claim 154, wherein saidantibody is a chimeric or a humanized antibody.
 158. The method of claim154, wherein said antibody, oligopeptide or organic molecule isdetectably labeled.
 159. The method of claim 154, wherein said testsample of tissue cells is selected from an individual suspected ofhaving a tumor.
 160. The method of claim 154, wherein said tumor isselected from the group consisting of lymphoma, myeloma and leukemia.161. The method of claim 154, wherein said cell is a hematopoietic cell.162. The method of claim 161, wherein said hematopoietic cell isselected from the group consisting of a lymphocyte, leukocyte, platelet,erythrocyte and natural killer cell.
 163. The method of claim 162,wherein said lymphocyte is a B cell or a T cell.
 164. The method ofclaim 162, wherein said lymphocyte is a cancer cell.
 165. The method ofclaim 164, wherein said cancer cell is selected from the groupconsisting of a lymphoma cell, a myeloma cell and a leukemia cell. 166.The method of claim 161, wherein said protein is more abundantlyexpressed by a hematopoietic cell as compared to a non-hematopoieticcell of said tumor.
 167. The method of claim 154, wherein said proteinhas: (a) the amino acid sequence selected from the group consisting ofthe amino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ IDNO: 4) and FIG. 6 (SEQ ID NO: 6); (b) the amino acid sequence selectedfrom the group consisting of the amino acid sequence shown in FIG. 2(SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lackingits associated signal peptide sequence; (c) an amino acid sequence of anextracellular domain of the polypeptide selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2),FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), with its associatedsignal peptide sequence; (d) an amino acid sequence of an extracellulardomain of the polypeptide selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO:4) and FIG. 6 (SEQ ID NO: 6), lacking its associated signal peptidesequence; (e) an amino acid sequence encoded by the nucleotide sequenceselected from the group consisting of the nucleotide sequence shown inFIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5);or (f) an amino acid sequence encoded by the full-length coding regionof the nucleotide sequence selected from the group consisting of thenucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3)and FIG. 5 (SEQ ID NO: 5).
 168. A method of detecting the presence ofcells in a sample suspected of containing said cells, said methodcomprising detecting said cells by detecting cells expressing apolypeptide comprising an amino acid sequence having at least 95% aminoacid sequence identity to: (a) the polypeptide having the amino acidsequence selected from the group consisting of the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ IDNO: 6); (b) the polypeptide having the amino acid sequence selected fromthe group consisting of the amino acid sequence shown in FIG. 2 (SEQ IDNO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), lacking itsassociated signal peptide; (c) an extracellular domain of thepolypeptide having the amino acid sequence selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2),FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6), with its associatedsignal peptide; (d) an extracellular domain of the polypeptide havingthe amino acid sequence selected from the group consisting of the aminoacid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) andFIG. 6 (SEQ ID NO: 6), lacking its associated signal peptide; (e) apolypeptide encoded by the nucleotide sequence selected from the groupconsisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1),FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or (f) a polypeptideencoded by the full-length coding region of the nucleotide sequenceselected from the group consisting of the nucleotide sequence shown inFIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5),said method comprising contacting said sample with an antibody,oligopeptide or organic molecule that binds to said protein anddetecting the formation of a complex between said antibody, oligopeptideor organic molecule and said protein, wherein the formation of a complexindicates the presence of said cell.
 169. The method of claim 168,wherein said antibody is a monoclonal antibody.
 170. The method of claim168, wherein said antibody is an antibody fragment.
 171. The method ofclaim 168, wherein said antibody is a chimeric or a humanized antibody.172. The method of claim 168, wherein said antibody, oligopeptide ororganic molecule is detectably labeled.
 173. The method of claim 168,wherein said sample is selected from an individual suspected of having atumor.
 174. The method of claim 173, wherein said tumor is selected fromthe group consisting of lymphoma, myeloma and leukemia.
 175. The methodof claim 168, wherein said cell is a hematopoietic cell.
 176. The methodof claim 175, wherein said hematopoietic cell is selected from the groupconsisting of a lymphocyte, leukocyte, platelet, erythrocyte and naturalkiller cell.
 177. The method of claim 176, wherein said lymphocyte is aB cell or a T cell.
 178. The method of claim 176, wherein saidlymphocyte is a cancer cell.
 179. The method of claim 178, wherein saidcancer cell is selected from the group consisting of a lymphoma cell, amyeloma cell and a leukemia cell.
 180. The method of claim 175, whereinsaid protein is more abundantly expressed by a hematopoietic cell ascompared to a non-hematopoietic cell of said tumor.
 181. The method ofclaim 168, wherein said protein has: (a) the amino acid sequenceselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6);(b) the amino acid sequence selected from the group consisting of theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO:4) and FIG. 6 (SEQ ID NO: 6), lacking its associated signal peptidesequence; (c) an amino acid sequence of an extracellular domain of thepolypeptide selected from the group consisting of the amino acidsequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG.6 (SEQ ID NO: 6), with its associated signal peptide sequence; (d) anamino acid sequence of an extracellular domain of the polypeptideselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4) and FIG. 6 (SEQ ID NO: 6),lacking its associated signal peptide sequence; (e) an amino acidsequence encoded by the nucleotide sequence selected from the groupconsisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1),FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO: 5); or (f) an amino acidsequence encoded by the full-length coding region of the nucleotidesequence selected from the group consisting of the nucleotide sequenceshown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) and FIG. 5 (SEQ IDNO: 5).
 182. A method of patient selection for treatment of tumors, saidmethod comprising detecting in tumor samples from prospective patientscells that have: (a) low or no expression of a polypeptide having anamino acid sequence having at least 95% amino acid sequence identity tothe polypeptide having the amino acid sequence selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2) andFIG. 6 (SEQ ID NO: 6) and significant expression of a second polypeptidehaving an amino acid sequence having at least 95% amino acid sequenceidentity to the polypeptide having the amino acid sequence shown in FIG.4 (SEQ ID NO: 4); (b) low or no expression of a polypeptide having anamino acid sequence having at least 95% amino acid sequence identity tothe polypeptide having the amino acid sequence selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2) andFIG. 6 (SEQ ID NO: 6), lacking its associated signal peptide, andsignificant expression of a second polypeptide having an amino acidsequence having at least 95% amino acid sequence identity to thepolypeptide having the amino acid sequence shown in FIG. 4 (SEQ ID NO:4), lacking its associated signal peptide; (c) low or no expression of apolypeptide having an amino acid sequence having at least 95% amino acidsequence identity to the polypeptide having the amino acid sequenceselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2) and FIG. 6 (SEQ ID NO: 6), with its associatedsignal peptide, and significant expression of a second polypeptidehaving an amino acid sequence having at least 95% amino acid sequenceidentity to the polypeptide having the amino acid sequence shown in FIG.4 (SEQ ID NO: 4), with its associated signal peptide; (d) low or noexpression of a polypeptide having an amino acid sequence having atleast 95% amino acid sequence identity to an extracellular domain of thepolypeptide having the amino acid sequence selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2) andFIG. 6 (SEQ ID NO: 6), lacking its associated signal peptide, andsignificant expression of a second polypeptide having an amino acidsequence having at least 95% amino acid sequence identity to anextracellular domain of the polypeptide having the amino acid sequenceshown in FIG. 4 (SEQ ID NO: 4), lacking its associated signal peptide;(e) low or no expression of a polypeptide having an amino acid sequencehaving at least 95% amino acid sequence identity to a polypeptideencoded by the nucleotide sequence selected from the group consisting ofthe nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1) and FIG. 5 (SEQID NO: 5) and significant expression of a second polypeptide having anamino acid sequence having at least 95% amino acid sequence identity toa polypeptide encoded by the nucleotide sequence shown in FIG. 3 (SEQ IDNO: 3); or (f) low or no expression of a polypeptide having an aminoacid sequence having at least 95% amino acid sequence identity to apolypeptide encoded by the full-length coding region of the nucleotidesequence selected from the group consisting of the nucleotide sequenceshown in FIG. 1 (SEQ ID NO: 1) and FIG. 5 (SEQ ID NO: 5) and significantexpression of a second polypeptide having an amino acid sequence havingat least 95% amino acid sequence identity to a polypeptide encoded bythe full-length coding region of the nucleotide sequence shown in FIG. 3(SEQ ID NO: 3), wherein the identification of said cells from said tumorsamples indicates the identification of a patient for treatment with anantibody, oligopeptide or organic molecule, conjugated to agrowth-inhibitory agent or a cytotoxic agent, said conjugated antibody,oligopeptide or organic molecule binds to said second polypeptide anddepends on internalization into said cells for effective treatment. 183.A method of patient selection for treatment of tumors, said methodcomprising detecting in tumor samples from prospective patients cellsthat have: (a) significant expression of a polypeptide having an aminoacid sequence having at least 95% amino acid sequence identity to thepolypeptide having the amino acid sequence selected from the groupconsisting of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2) andFIG. 6 (SEQ ID NO: 6) in a minority of the cells or in a population ofcells distinct from cells that with significant expression of a secondpolypeptide having an amino acid sequence having at least 95% amino acidsequence identity to the polypeptide having the amino acid sequenceshown in FIG. 4 (SEQ ID NO: 4); (b) significant expression of apolypeptide having an amino acid sequence having at least 95% amino acidsequence identity to the polypeptide having the amino acid sequenceselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2) and FIG. 6 (SEQ ID NO: 6), lacking its associatedsignal peptide, in a minority of the cells or in a population of cellsdistinct from cells with significant expression of a second polypeptidehaving an amino acid sequence having at least 95% amino acid sequenceidentity to the polypeptide having the amino acid sequence shown in FIG.4 (SEQ ID NO: 4), lacking its associated signal peptide; (c) significantexpression of a polypeptide having an amino acid sequence having atleast 95% amino acid sequence identity to the polypeptide having theamino acid sequence selected from the group consisting of the amino acidsequence shown in FIG. 2 (SEQ ID NO: 2) and FIG. 6 (SEQ ID NO: 6), withits associated signal peptide, in a minority of the cells or in apopulation of cells distinct from cells with significant expression of asecond polypeptide having an amino acid sequence having at least 95%amino acid sequence identity to the polypeptide having the amino acidsequence shown in FIG. 4 (SEQ ID NO: 4), with its associated signalpeptide; (d) significant expression of a polypeptide having an aminoacid sequence having at least 95% amino acid sequence identity to anextracellular domain of the polypeptide having the amino acid sequenceselected from the group consisting of the amino acid sequence shown inFIG. 2 (SEQ ID NO: 2) and FIG. 6 (SEQ ID NO: 6), lacking its associatedsignal peptide, in a minority of the cells or in a population of cellsdistinct from cells with significant expression of a second polypeptidehaving an amino acid sequence having at least 95% amino acid sequenceidentity to an extracellular domain of the polypeptide having the aminoacid sequence shown in FIG. 4 (SEQ ID NO: 4), lacking its associatedsignal peptide; (e) significant expression of a polypeptide having anamino acid sequence having at least 95% amino acid sequence identity toa polypeptide encoded by the nucleotide sequence selected from the groupconsisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1) andFIG. 5 (SEQ ID NO: 5), in a minority of the cells or in a population ofcells distinct from cells with significant expression of a secondpolypeptide having an amino acid sequence having at least 95% amino acidsequence identity to a polypeptide encoded by the nucleotide sequenceshown in FIG. 3 (SEQ ID NO: 3); or (f) significant expression of apolypeptide having an amino acid sequence having at least 95% amino acidsequence identity to a polypeptide encoded by the full-length codingregion of the nucleotide sequence selected from the group consisting ofthe nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1) and FIG. 5 (SEQID NO: 5) in a minority of the cells or in a population of cellsdistinct from cells with significant expression of a second polypeptidehaving an amino acid sequence having at least 95% amino acid sequenceidentity to a polypeptide encoded by the full-length coding region ofthe nucleotide sequence shown in FIG. (SEQ ID NO: 3), wherein theidentification of said cells from said tumor samples indicates theidentification of a patient for treatment with an antibody, oligopeptideor organic molecule, conjugated to a growth-inhibitory agent or acytotoxic agent, said conjugated antibody, oligopeptide or organicmolecule binds to said second polypeptide and depends on internalizationinto said cells for effective treatment.